Detailed descriptions about quarantine and regulations, new laws, and new techniques are using in plant quarantine for the detection of plant pathogens are described

1. QUARANTINE REGULATION AND THE
IMPACT OF MODERN DETECTION
METHODS
1
C.GUNASEELI
II-M.Sc.,(Ag.) - Plant
pathology
Annamalai University

2. Plant Quarantine
2
• Italian word
• Quarantine = 40 days
• Quarantine regulations are formulated by goverments to reduce the chances of
pests or diseases being introduced on articles imported from foreign countries.
Sanhitha Sharma et al. 2007

3. 3
REGULATORY MEASURES
• To prevent the introduction of exotic pests and diseases into the
country.
• To suppress or prevent the spread of pests and diseases in localized
area with in a state or union territory
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4. HISTORY
 The first plant quarantine was passed - 1660
 Embargo passed in Germany - 1873
 UK destructive insect act - 1877
 Federal quarantine service established in Australia - 1909
 Federal quarantine act in USA - 1912
 First Plant Quarantine and Fumigation Station Dec 25,1951
 National Bureau of Plant Genetic Resource (NBPGR) August, 1976
 Division of Plant Quarantine with Entomology,
Plant Pathology and Nematology sections 1978
 Plants, Fruits and Seeds (Regulation of import into India)
PFS October , 1988
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PLANT PROTECTION IN INDIA -MILESTONE
Destructive Insects and Pests Act 1914
Locust Warning Organization 1939
Directorate of Plant Protection, Quarantine and Storage 1946
National Institute of Plant Health Management 1966
Implementation of Insecticides Act ,1968 1975
Integrated Pest Management (Central IPM Centres ) 1992
Plant Quarantine Order 2003
Online Registration of Pesticides 2010
Online PQ Services (PQIS) 2011

6. DOMESTIC QUARANTINE
• State to state enforcement
Restricts the inter-state movement of pests/diseases:
Sanjose scale (Kashmir)
Banana bunchy top virus (Assam Kerala, TN and W.B.)
Banana mosaic virus (Maharashtra and Gujarat )
Potato cyst nematode (Nilgiri district of TN)
Potato wart (Darjeeling district of West Bengal)
Apple scab (JK and HP)
Chand et al. 2017
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7. LAWS IN TAMIL NADU
• The Madras Agricultural Pests and Diseases Act, 1919.
• The Tamil Nadu Agricultural Pests and Diseases (Amendment) Act, 1982
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8. INTERNATIONAL QUARANTINE
• Country to country enforcement
Prohibited import crops in India
Moko wilt of Banana Race 2 (Central and South America ,Philipines)
Cassava bacterial blight-American strains, cassava viruses (Africa & South
America)
Chestnut Blight of Canker (North America)
Potato smut & Potato viruses (South America)
Sugarcane Fiji virus (Fiji, Australia, Philippines and Indonesia)
Yam mosaic virus (West Africa and Caribbean )
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10. S.No. Diseases Introduced Year
in from
1. Late blight of potato Europe S. America 1830
2. Grape Phylloxera France USA 1845
3. Golden nematode of potato USA, Mexico Europe 1881
4. Chestnut blight USA Asia 1904
5. Citrus canker USA Asia 1907
6. Blister rust of pine USA Europe 1910
7. Fire blight of apple New Zealand N-America 1919
8. Bacterial canker of Tomato UK USA 1942
9. Dutch elm USA Holland 1928-1930
Diseases which have been introduced world-wise
(Rai Vijay Lakshmi et al. 2014)
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DISEASES INTRODUCED INTO INDIA FROM
OTHER COUNTRIES
S.
o
DISEASES NATIVE PLACE YEAR
1. Coffee rust ,
BBTV
Srilanka 1879
1940
2. Late blight of potato England 1883
3. Flag smut oF wheat Australia 1906
4. Downy Mildew of grapes Europe 1910
5. DM of maize Java 1912
6. Fire blight of Pear , Crown gall of Apple and
pear
England 1940
7. Potato wart Netherland 1953
8. Golden nematode of Potato Europe 1961
9. San jose scale of apple Italy 1900
Chand et al. 2017

12. NATIONAL PLANT PROTECTION ORGANISATION
AGRICULTURE MINISTER
SECRETARY (AGRI& COOP)
ADDITIONAL SECRETARY
JOINT SECRETARY (PLANT PROTECTION)
PLANT PROTECTION ADVISER TO THE GOVERNMENT OF
INDIA
Plant
Protection
(IPM)
Plant Quarantine Locust control
Pesticide
Registration
and Quality
Control
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13. NATIONAL PLANT PROTECTION ORGANISATION
Plant Protection
(IPM)
Plant
Quarantine
(PQ)
Locust
Control
Pesticide Registration
& Quality Control
5 Regional Central
IPM Centres
7 Regional PQ
Stations
66 PQSs
Locust
Warning
Organization
10 LWO Offices
1. Sect. for
Registration of
Pesticides
2. Central
Insecticide Lab
13
Plant protection advisor
26 CIPMCS

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PLANT QUARANTINE -SET UP
73 Plant quarantine station
Plant quarantine station under RPQS, Mumbai – 9
Plant quarantine station under RPQS, Kandla -10
Plant quarantine station under RPQS, Chennai-12
Plant quarantine station under RPQS, Bengaluru-10
Plant quarantine station under RPQS, Kolkata -17
Plant quarantine station under RPQS, News Delhi -10
Plant quarantine station under RPQS, Amristar - 5

15. Plant Quarantine - Set up
 94 entry points
 Land Frontier 24
 International Airports 24
 Sea Ports 46
Foreign Post offices 11
 Phytosanitary certification
 198 Government Officers from Central/State/UT are
authorized
 Fumigation & Treatment
 375 Pest Control Agencies accredited
 169 Forced Hot Air Treatment Providers accredited
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17. PHYTOSANITORY CERTIFICATE
What is a Phytosanitary Certificate?
A Phytosanitary Certificate is an official document that indicates the exporting material is free from
pest and diseases.
(http://www.agriculture.gov.ie/farmingsectors/planthealthtrade/)
When should a Phytosanitary Certificate be required?
Importing Countries should only require phytosanitary certificates for regulated articles. These
include
commodities such as plants, bulbs and tubers, or seeds for propagation, fruits and vegetables,
cut flowers and branches, grain and growing medium.
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19. Guidelines for import of Plant material
• Import from a country where the pathogen(s) is absent.
• Import from a country with an efficient plant quarantine service, so that
inspection and treatment is done.
• Obtain Planting material from the safest known source within the selected
country.
• Obtain non-treated seeds so that detection of seed borne pathogens is facilitated
• Obtain clean, healthy-looking seeds of type of impurities.
• Obtain an official certificate of freedom from pests and diseases from the
exporting country.
• Import the smallest possible amount of planting material; the smaller
the amount, the less the chance of its carrying infection.
• It will also simplify post entry inspection.
• Inspect material carefully on arrival and treat.
• If other precautions are not adequate, subject the material to intermediate or
post entry quarantine.
• Salvage infected seeds.
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20. • Visual inspection
• Isolation on growth medias
• Bioassays of sensitive indicators of
inoculated for specific symptoms
• Microscopy
• Serology
• Molecular methods
• Polymerase chain reaction (PCR)
• Reverse transcriptase- PCR (RT- PCR)
• Quantitative PCR (q - PCR) and
• Nucleic acid lot assays.
INSPECTION PROCEDURES IN QUARANTINE STATION
20

21. List of Viral pathogens intercepted in
germplasm importing
VIRUS HOST
GEOGRAPHICAL
DISTRIBUTION
Ficus Mosaic
Virus
Ficus sp. North America
Grape fan leaf
virus
Vitis vinifera (p) South Pacific Asia
Mosaic virus Jasminum spp,
Hibiscus cooperi (P)
Asia, Europe
Pea Mosaic
virus
Pisum sativum (S) South Pacific Asia
Orchid virus Orchid (P) Asia
Spotted wilt
virus
Chrysanthemum sp.
(p)
Europe
Tobacco
mosaic virus
Dahelia variabilis (T) Europe, North America
S – Seed, P- Plant , T- tubers
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22. List of fungal pathogens intercepted
in germplasm importing
Fungal pathogen Host Geographical
distribution
Alternaria helianthi Helianthus annus (S)
Sorghum sp.
North America
Ascochyta gossypii Gossypium sp. (s) Africa
Ascochyta rabiei Arachia hypogeal (s) Africa
Botrytis fabae Vicia faba (s) Asia
Cercospora dolichi Betal leaf North America
Drecheslera spicifera Thugapli catu (s) North America
Phoma coccicola Cocus nucifera(s) Asia
Fusarium avenaceum Celosia sp. North America
Septoria gladioli Gladiolus sp. (s) Europe
Drechslera maydis Sorghum sp. (S), Spinaua
oleracea (s)
Europe, North America,
South America
S – Seed, P- Plant , t- tubers Usha DEV et al.
12
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23. List of Bacterial pathogens intercepted in
germplasm importing
Bacteria Host Geographical
Distributiom
Agrobacterium Rosa sp. (p) Europe
Corynebacterium
michiganense
Lycopersicon
esculentum(s)
North America
Erwinia carotovora Solanum tuberosum (t) Europe, North
America
Erwinia herbicola Helianthus annus (s) Europe
Pseudomonas maculicola Brassica oleracea var.
capitata (S)
Europe, North
America
Pseudomonas marginatum Gladiolus sp. (b) Europe
Pseudomonas pisi Pisum sativum (S) North America,
Europe
Pseudomonas syringae Cucumis Sativus (S) North America
Xanthomonas campestris Citrus sp. (f) Asia
S – Seed, P- Plant , t- tubers, b- bulbs (Rai vijay Lakshmi et al. 2014)
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NBPGR has listed the exotic diseases
1.Peanut stripe virus in 1987
2.Banana bract virus & Banana streak virus in 1995
3.Bemisia tabaci biotype B in 1999
4.Coconut mite in 1995

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26. WHY MODERN TECHNIQUES ARE
REQUIRED IN QUARANTINE .?
• Conventional diagnostic techniques are time consuming, also need more labour
and skill users.
Newly entered pathogens in India
Fungi – Wheat blast Motalleb KA et al. 2019
Bacteria – all races of Ralstonia solanacearum
Virus – Grape vine leaf roll virus Surender kumar et al. 2013
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27. MODERN DETECTION METHODS
AND TECHNIQUES
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28. 1. PCR ( POLYMERASE CHAIN REACTION )
2. LAMP ( LOOP MEDIATED ISOTHERMALAMPLIFICATION )
3. NGS ( NEXT GENERATION SEQUENCING )
4. PYROSEQUENCING
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DETECTION METHODS

29. PCR – POLYMERASE CHAIN REACTION
• PCR technique is used to amplify the target DNA invitro. It is otherwise
known as thermal cycler
TYPES
• Conventional PCR
• Reverse transcription PCR(RT PCR)
• Real time PCR ( qPCR)
• Multiplex PCR
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30. PCR (POLYMERASE CHAIN REACTION)
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32. RT- PCR
• Important limitations of other PCR
types are inability to differentiate
between dead and living fungi. But
RT- PCR can do…
• Process - RNA reverse transcribed to
cDNA and then amplified by any PCR
based methods
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34. • Quantify the Fusarium ear blight (Fusarium
graminearum) in cereals such as wheat, rye,
barley, oat and maize
(Brown et al.2011)
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35. REAL TIME PCR (q-PCR)
• Amplification of target sequence can be quantified at each PCR
cycle, an amplification is monitored pictographically in monitor.
• Eliminates the post PCR steps, also eliminates carcinogenic
chemicals (Ethidium bromide and radioactive isotopes, UV
radiation)
• Reduce the risk of sample contamination, and check the cell
viability
(Capote et al. 2012)
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37. • There are currently 4 different fluorescent techniques
- SYBR green dye based detection technique
- TaqMan probes
- Fluorescent resonance energy transfer (FRET)
- Molecular beacons
(Schaad and Frederick 2002, Mackey et al. 2002)
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38. Used for the identification and quantification of disease causing fungi
Aspergillus versicola
Cladosporium cladosporides
Stachybotrytis chartrum and
Alternaria alternata
(Black 2009)
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39. MULTIPLEX PCR
• Simultaneously detect multiple pathogens in single reaction. More
than one set of primers are used
• Reduce the total detection time.
• Used for the simultaneous detection of fungal pathogens like F.
oxysporium, P. nicotianae and P. cactorum
(Cho et al. 2016)
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It Quantifies RNA viruses and Pseudomonas savastanoi pv. savastanoi
affecting olive tress.
(Bertolini et al. 2003)

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41. LAMP (LOOP MEDIATED ISOTHERMAL
AMPLIFICATION)
• Amplifies nucleic acid under isothermal conditions with high specificity for
isolation of DNA at single temperature
(Tsui et al. 2011)
• Isothermal amplification is carried out at a constant temperature (60 – 65oC)
• Typically, 6 different primers are used to identify 8 distinct regions on the target
gene
(Fauruddin et al. 2011)
• Use BST polymerase for high strand displacement activity.
• It may be combined with a reverse transcription step to allow the detection of RNA
• 10 times more sensitive than conventional PCR (Ren et al. 2009)
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43. • We can diagnose diseases in Ascochyta blight (Ascochyta rabiei)
(Das et al. 2012)
• Detect the presence of thermo-dependent dimorphic fungus
Paracoccdioides brasiliensis
(Endo et al. 2004)
• Penicillium marneffei (Sun et al. 2010)
• Primary blue stain fungus (Villari et al. 2013)
• Erwinia amylovora in pear and apple (Temple et al. 2008)
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BY USING LAMP

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46. NEXT GENERATION SEQUENCING
(NGS)
 NGS technology is embraced in Plant Pathology.
 Used to detect multiple pests in different kingdoms and down to strain in single test (Wu et al. 2015)
 Detect very low titer organisms (Stobbe and Roossinck et al. 2011)
 Cost is reduced and tool for plant quarantine and protocols
(Adams et al. 2009)
 NGS can be as sensitive as graft indexing onto indicator plants for
detection of known viruses of grapevine.
(Rwahnih et al. 2015)
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48. USING NGS
• Grapevine red blotch associated virus (Sudarshana et al. 2015)
• Detection of new Luteovirus in nectarine germplasm from France after clearing post
entry quarantine
(Bag et al. 2015)
• NGS used to develop draft genomes of Calonectria henricotiae and C.
pseudonaviculata (blight on Sarcococca) fungal pathogens causing boxwood blight
(Malapi-Wight et al. 2016)
• Detection of sugarcane viruses in quarantine programs
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51. HOW NGS CAN BE USED IN QUARANTINE
• Rapidly sequence whole genomes
• Pathway analysis
• Broad Dynamic Range for Expression Profiling
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53. PYROSEQUENCING
• DNA sequencing technology based on the sequencing by synthesis principle
Polymerase I - (DNA)n + dNTP (DNA)n+1 + PPi
ATP - Sulfurylase – Ppi + APS ATP + SO4
2-
Luciferses - Lucifersase – luciferin + ATP Light
Apyrase - ATP AMP + 2Pi
(Nyren et al. 1987)
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54. PRINCIPLE
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60. OTHER DETECTION
TECHNIQUES INCLUDES
• RFLP
• RAPD
• AFLP
• Nested PCR
• Bio PCR
• Microrraays
• ELISA
• FISH hybridisation
• NASBA
• SAGE (serial analysis
gene expression)
• Blotting techniques
• DNA barcoding
• Insitu hybridization
• Luminex
• Biosensor
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61. References –
• Afshin Ahmadian, Maria Ehn, Sophia Hober (2005). Pyrosequencing: History, biochemistry and
future. Science and direct. 363 (2006): 83 – 94.
• Ahmed Hadidi, Ricardo Flores, Thierry Candresse and Marina Barba (2016). Next-Generation
Sequencing and Genome Editing in Plant Virology. Frontiers in Microbiology. 7: 1 -12.
• Kalyan K. Mondal and V. Shanmugam (2013). Advancements in the diagnosis of bacterial plant
pathogens: An overview. Biotechnology and Molecular Biology Review. 8 (1): 1-11.
• Rai Vijay Laxmi, Geetanjaly and Sharma Preeti (2014). Plant Quarantine: An Effective Strategy of
Pest Management in India. Res. J. Agriculture and Forestry Sci. 2 (1): 11 – 16.
• Shivan and Pandey (2010). hybridoma technology for production of monoclonal antibodies.
International Journal of Pharmaceutical Sciences Review and Research. 1(2): 88 – 94.
• Sidra Aslam, Aisha Tahir, Muhammad Farhan Aslam, Muhammad Waqar Alam, Arshad Ali Shedayi &
Sehrish Sadia (2016). Recent advances in molecular techniques for the identification of
phytopathogenic fungi – a mini review. Journal of Plant Interactions. 12(1): 493 – 504.
• Platforms for the detection of plant diseases and –pests (WAGENINGEN university and research)
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