includes quarantine details and advanced equipment's in the the quarantine and in the field of biology

1. ANNAMALAI UNIVERSITY
FACULTY OF AGRICULTURE
DEPARTMENT OF PLANT PATHOLOGY
Manuscript on
QUARANTINE REGULATION AND THE IMPACT OF MODERN DETECTION
METHODS
Submitted by
N. H. SHANKAR REDDY
Reg no – 091712
IInd
M.Sc., PlantPathology

2. QUARANTINE REGULATION AND THE IMPACT OF MODERN DETECTION
METHODS
Quarantine –
 The term quarantine is derived from ‘Latin’ word which means ‘40days period’.
 All activities which are designed to prevent the introduction and spread of pest/
diseases to ensure official control (legal enforcement isolation) to ensure bio security
is termed as ‘Quarantine’
HISTORY
 The first plant quarantine was passed - 1660
 First Embargo passed in Germany - 1873 (to control
Potato Colorado beetle)
 UK destructive insect act - 1877 (to control
Colorado beetle)
 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
PLANT PROTECTION IN INDIA – MILESTONES
 Destructive and insects Pest act - 1914
 Locust warning organization - 1939
 Directorate of Plant Protection, Quarantine and Storage - 1946
 National institute of Plant Health Management - 1966
 Implementation of insecticide act, 1968 - 1975
 Integrated pest management (central IPM centres) - 1992

3.  Plant quarantine order - 2003
 Online registration of pesticides - 2010
 Online PG services (PQIS) - 2011
Quarantine is equipped into 3 divisions
1. Domestic quarantine
2. International quarantine
3. Embargo
1. Domestic quarantine -
Restriction to entry of plant and plant related material form one state to another state
(which is associated with sate machinery production)
 Sanjose scale – which asses the losses which was caused by insects.
 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)
These infected materials which are prohibited to transport to other states for trade and
export.
Laws which was enforced in Tamil Nadu
 The Madras Agricultural Pests and Diseases Act, 1919.
 The Tamil Nadu Agricultural Pests and Disease (Amendment) Act, 1982
2. International quarantine -
Legal restriction on plant and plant related products between on e country to another
country to ensure pest and diseases free materials.
3. Embargo –
Official ban on trade or other commercial activity with a particular country.
DISEASES WHICH HAVE BEEN INTRODUCED WORLD-WISE
Sl. no Diseases Introduced Year
from in
1 Late blight of potato S. America Europe 1830
2 Grape Phylloxera USA France 1845
3 Golden nematode of potato Europe USA, Mexico 1881
4 Chestnut blight Asia USA 1904

4. 5 Citrus canker Asia USA 1907
6 Blister rust of pine Europe USA 1910
7 Fire blight of apple N. america New Zealand 1919
8 Bacterial canker of Tomato USA UK 1942
9 Dutch elm Holland USA 1928-1930
.
PESTS AND DISEASES INTRODUCED IN INDIA FROM OTHER COUNTRIES
Sl. no Pests Native place Year of
introduction
1 Coffee rust (Hemileia vastatrix) Sri Lanka 1879
2 Late blight of potato (Phytophthora infestans) England 1883
3 Flag smut of wheat (Urocystis tritici) Australia 1906
4 Downy mildew of grapes (Plasmapara viticola) Europe 1910
5 Rust if chrysanthemum (Puccinia carthami) Japan/ Europe 1904
6 Downy mildew of cucurbits (P. cubensis) Sri Lanka 1918
7 Downy mildew of maize (S. Philippiensis) Java 1912
8 Foot rot of rice (Fusarium moniliforme) South east
Asia
1930
9 Black rot of crucifers (Xanthomonas compestris) Java 1929
10 Leaf spot of sorghum (Phyllochora sorghi) South Africa 1934
11 Powdery mildew of rubber (Oidium heveae) Malaysia 1938
12 Black shank of tobacco (P. nocotianae) Holland 1938
13 Fire blight of pear (Erwinia amylovora) England 1940
ORGANIZATIONAL SETUP IN INDIA

6. Plant Quarantine - Set up
 35 Plant Quarantine Stations
 Land Frontier 13
 International Airports 11
 Sea Ports 11
 Phytosanitary certification
 151 Government Officers from Central/State/UT are authorized
 Fumigation & Treatment
 375 Pest Control Agencies accredited
 169 Forced Hot Air Treatment Providers accredited
PHYTOSANITORY CERTIFICATE
A Phytosanitary Certificate is an official document
- issued by the Horticulture and Plant Health Division of the Department of
Agriculture, Food and the Marine to indicate that consignments of plants, plant
products or other regulated articles meet specified phytosanitary import meets the
requirements of the National Plant Protection Organisation (NPPO) of the Importing
Country
(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.

7. Certification programme includes
• visual inspection of germplasm
• 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.
Guidelines for import of Germplasm
 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

8.  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
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
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

9. 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
List of Bacterial pathogens intercepted in germplasm importing
Bacteria Host Geographical Distributiom
Agrobacterium tumefaciens 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 pv. citri Citrus sp. (f) Asia
Plant Quarantine - New Initiatives
• 22 new PQ Stations to service additional entry points being set up
• ISO Certification for PQ Stations for quality service being obtained
• Online Plant Quarantine services launched in January 2011
(www.plantquarantineindia.nic.in)
• Establishment of National Agricultural Biosecurity System (NABS) on the anvil with
new legislation
WHY MODERN TECHNIQUES ARE REQUIRED IN QUARANTINE .?

10. • Conventional diagnostic techniques are time consuming, also need more labour and
skill users.
• To prevent the entry of new pest/ alien pests
Eg – Sunflower necrosis virus
Newly entered pathogens in India
Fungi – Wheat blast
Bacteria – all races of Ralstonia solanacearum
Virus – Grape vine leaf roll virus
MODERN DETECTION METHODS AND TECHNIQUES
1. PCR (Polymerase chain reaction)
2. MONOCLONAL ANTIBODIES (HYBRIDOMA TECHNOLOGY)
3. PYROSEQUENCING
4. NEXT GENERATION SEQUENCING (NGS)
5. LAMP (Loop mediated isothermal amplification)
6. BIOINFORMATICS
Types of PCR
A. PCR
B. Reverse transcription PCR (RT-PCR)
C. Real time PCR (q PCR)
D. Multiplex PCR

11. A. PCR (Polymerase chain reaction)
Which involves in 3 steps
Denaturation (92-94oC )
Annealing (55-65oC)
Extension (72 oC)
B. Reverse transcription PCR (RT-PCR)
 Since the discovery some of the changes have been expanded utility an diagnostic
capability of PCR in many biological and medical fileds (Tang et al., 1997)
 Important limitations of other PCR types is their inability to differentiate between
dead and living cells. This limitation can be overcome by the discovery of RT-
PCR
 In this process, firstly, the RNA is reverse transcribed into cDNA by random
primers and RT enzyme and then amplified by any PCR based methods.
One step and two step variants are available in RT- PCR

12.  The quantification
of mRNA using RT-PCR can be
achieved as either a one-step or a
two-step reaction.
The difference between the two
approaches lies in the number of
tubes used when performing the
procedure. In the one-step approach,
the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube.
On the other hand, the two-step reaction requires that the reverse transcriptase
reaction and PCR amplification be performed in separate tubes. The one-step
approach is thought to minimize experimental variation by containing all of the
enzymatic reactions in a single environment.
 RT- PCR used to Quantify the Fusarium ear blight (Fusarium graminearum) diseases
in cereals such as wheat, rye, barley, oat and maize.
C. Real time PCR (q PCR)
 Real time PCR is the most advanced version of PCR
 Where the amplification of target sequence can be quantified after each PCR cycle
and amplification is pictographically displayed through an attached monitor
 It gains popularity not only eliminating post PCR steps and including
electrophoresis, but also eliminates the exposure of carcinogenic chemicals,
ethidium bromide, radioactive isotopes and UV radiation.
 It also reduced the risk of contamination.
There are currently 4 different fluorescent techniques used to detect amplicons
- SYBR green dye based detection technique
- TaqMan probes
- Fluorescent resonance energy transfer (FRET)
- Molecular beacons
Used for the identification and quantification of disease causing fungi
o Aspergillus versicola
o Cladosporium cladosporides
o Stachybotrytis chartrum and
o Alternaria alternata
D. Multiplex PCR
 Useful for the simultaneous detection of multiple pathogens in single reaction.
more than one set of primers are used.

13.  Different fragments that are specific to the target pathogenic fungi were
simultaneously amplified, and detected based on the molecular size on the
agarose gel.
 To avoid pifull, primers must be designed carefully along with relative
concentration and annealing temperature.
 Now a day’s padlock probes (PLFs) are used in the multiplex technique for the
identification of fungi.
 Used for the simulations detection of fungal pathogens like F. oxysporium, P.
nicotianae and P. cactorum
 It also Quantifies four RNA viruses and Pseudomonas savastanoi pv.
Savastanoi affecting olive trees.
2. HYBRIDOMA TECHNOLOGY
 Hybridomas are cells that have been engineered to produce a desired antibody in large
amounts, to produce monoclonal antibodies.
 Hybridoma technology was discovered in 1975 by two scientists, Georges Kohler of
West Germany and Cesar Milstein of Argentina (now working in U.K.), who jointly
with Niels Jerne of Denmark (now working in Germany) were awarded the 1984
Noble prize for physiology and medicine
 A hybridoma, which can be considered as a harry cell, is produced by the injection of
a specific antigen into a mouse, procuring the antigen-specific plasma cells (antibody-
producing cell) from the mouse's spleen and the subsequent fusion of this cell with a
cancerous immune cell called a myeloma cell.
 The advantage of this process is that it can combine the qualities of the two different
types of cells; the ability to grow continually, and to produce large amounts of pure
antibody.
 HAT medium (Hypoxanthine Aminopetrin Thymidine) is used for preparation of
monoclonal antibodies.
 Fused cells are incubated in the HAT (Hypoxanthine Aminopetrin Thymidine)
medium. Aminopterin in the myeloma cells die, as they cannot produce nucleotides
by the de novo or salvage medium blocks the pathway that allows for nucleotide

14. synthesis. Hence, unfused D cell die. Unfused B cells die as they have a short life
span. Only the B cell-myeloma hybrids survive, since the HGPRT gene coming from
the B cells is functional. These cells produce antibodies (a property of B cells) and are
immortal (aproperty of myeloma cells).
Advantages
• combine the qualities of the two different types of cells; the ability to grow
continually, and to produce large amounts of pure antibody
• Ability to produce MAbs for indefinite time period my cryopreservation of
hybridomas or unlimited periods
Disadvantages
• Production of mAbs is both expensive and time-consuming
• Proteins expressed in bacteria often fail to recognize protein in planta.
• in the luteovirus group, tests with Barley yellow dwarf virus (BYDV) have
shown high cross-reactivity between different type/ strains.
Examples -
• Raspberry bushy dwarf virus
• Potyvirus – genera specific monoclonal antibody have developed for first-
screen.
• Used to detect plants systemically infected with Cowpea severe mosaic virus
(CPSMV) and Papaya lethal yellowing virus (PLYV)
3. PYROSEQUENCING
 Pyrosequencing is a DNA sequencing technology based on the sequencing by-
synthesis principle.

15.  The real time monitoring of DNA synthesis, the sequencing by-synthesis principle,
was first described in 1985.
 In 1987, P. Nyren described how DNA polymerase activity can be monitored by
bioluminescence. Recently fluorescently labeled nucleotides have been used for
sequencing by synthesis.
 In the early days this sequence technology utilized six different sequential columns
with immobilized enzymes to pass the nucleotides through upon each base addition.
Ten years later, the pyrosequencing DNA sequencing method was presented enabling
faster bioluminometric real-time sequence determination in solution.
(Depending on the intensity of light we can determine the type of nucleotide added
and sequenced)
The pyrosequencing principle
 The 4 enzymes included in the pyrosequencing system
DNA Polymerase I ,
ATP sulfurylase ,
Luciferase and
Apyrase
 The reaction mixture also contains the enzyme substrates adenosine phosphosulfate
(APS), d-luciferin and the sequencing template with an annealed primer to be used as
starting material for the DNA polymerase.
 The four nucleotides are added one at a time, iteratively, in a cyclic manner and a
CCD camera detects the light produced.
 The first reaction, the DNA polymerization, occurs if the added nucleotide forms a
base pair with the sequencing template and thereby is incorporatedinto the growing
DNA strand.
(DNA)n + dNTP (DNA)n+1 + PPi
 The inorganic pyrophosphate, PPi, released by the DNA polymerase serves as
substrate for ATP Sulfurylase, which produces ATP:
PPi+ APS ATP + SO4
2-
 Through the third and fourth reactions, the ATP is converted to light by Luciferase
and the light signal is detected. Hence, only if the correct nucleotide is added to the
reaction mixture, light is produced.
Lucifersase – luciferin + ATP Lucifersase – luciferin – AMP+Pi
Luciferase - luciferin - AMP O2 Luciferase + oxyluceferin + AMP + CO2 + light
 Apyrase removes unincorporated nucleotides and ATP between the additions of
different bases.
ATP AMP + 2Pi
dNTP dNMP + 2Pi

16. 4. NEXT GENERATION SEQUENCING
 Includes several high throughput DNA sequencing technologies capable of
sequencing millions of molecules simultaneously
 Sequence numerous genes at a time or all DNA in a sample
 It is used to detect pest from different kingdoms and strains in a single test.
 Used to detect known knowns samples (Visible symptoms, with known pathogen),
known unknowns samples (Visible symptoms, but unknown pathogen), unknown
unknowns samples (Cryptic symptoms, unknown pathogen).
How NGS can improve plant protection
o Improved detection of pests
 Detection of unknown and cryptic pathogens
 Faster method to detect pests and certify that post-therapy material is free from
pests
o Certified plant material for export
o Pest identification
 Identify novel or unknown pests without pre-existing sequence knowledge
 Quickly identify DNA sequences that distinguish species, strains to design new
diagnostic tests
o Pathway analysis
 Rapidly sequence thousands of DNA regions to identify pest origin
Uses of NGS in plant quarantine
• Detect Grapevine red blotch associated virus
• Detection of new Luteovirus in nectarine germplasm from France after clearing post
entry quarantine
• NGS used to develop draft genomes of Calonectria henricotiae and C.
pseudonaviculata (blight on Sarcococca) fungal pathogens causing boxwood blight
• Detection of sugarcane viruses in quarantine programs

17. •
5. LAMP (LOOP MEDIATED ISOTHERMAL AMPLIFICATION)
 This technique was develiped by Notomi et al in 2000
 It amplifies the targeted nucleic acid under the isothermal conditions with the high
specificity. LAMP does not require a thermal cycler to produce the temperature
changes instead it requires single temperature for DNA amplification.
 Bst DNA polymerase and a set of four primers that consist of two inner and two outer
primers are used, which recognize total six unique sequences on the targeted DNA.
Two inner primer are referred to as forward inner primer (FIP) and backward inner
primer (BIP).
 One of the inner primers is used to begin the LAMP reaction while the other for self-
priming. LAMP reaction is processed in a heat block or water bath at 65°C for 1 h.
Then amplification product is detected by using the dye SYBR Green 1 as well as
by electrophoresis.
 Final product has many inverted repeats of targeted sequence which exhibit
cauliflower-like structure with multiple loops. LAMP is 10 times more sensitive and
accurate than the conventional PCR. Due to high-amplification efficiency, up to 1039
copies of a target part can be attained in less than 1 h of incubation.

18. By using LAMP
o We can diagnose diseases in Ascochyta blight (Ascochyta rabiei)
Detect the presence of thermo-dependent dimorphic fungus Paracoccdioides
brasiliensis
o Penicillium marneffei
o Primary blue stain fungus
o Erwinia amylovora in pear and apple.
o Through this technology, the fungus Magnaporthe oryzae is identified, which
causes rice blast disease in rice
o The fungus Verticillium dahliae is identified in tomato, which causes Vascular
wilt disease
6. BIOINFORMATICS
 Bioinformatics is an interdisciplinary field that develops methods and software
tools for understanding biological data. Bioinformatics combines biology, computer
science, information engineering, mathematics and statistics to analyze and interpret
biological data
 Bioinformatics to identify signature structure or motifys (nucleotide or amino-acid
sequence)
 Bioinformatics also tries to understand the organisational principles within nucleic
acid and protein sequences, called proteomics.
 The 2 important models in bioinformatics are

19. BLOSUM (Blocks substitution matrix)
 It is a substitution matrix used for sequence alignment of proteins.
 BLOSUM matrices are used to score alignments between evolutionarily divergent
protein sequences.
 They calculated a log-odds score for each of the 210 possible substitution pairs of the
20 standard amino acids.
BLASTx (basic local alignment search tool)
 It is an algorithm for comparing primary biological sequence information, such as
the amino-acid sequences of proteins or
the nucleotides of DNA and/or RNA sequences..
 Different types of BLASTs are available according to the query sequences.
 BLAST is one of the most widely used bioinformatics programs for sequence
searching.
 This emphasis on speed is vital to making the algorithm practical on the huge
genome databases currently available, although subsequent algorithms can be even
faster
 Before BLAST, FASTA was developed by David J. Lipman and William R.
Pearson in 1985.
BIOSENSORS
• Used for the detection of an analyte, that combines a biological component with
a physicochemical detector i.e. tissue, microorganisms, organelles, cell
receptors, enzymes, antibodies, nucleic acids, also used to detect toxic metabolites
such as mycotoxins.

20. Other detection techniques includes
RFLP (restriction fragment length polymorphism)
RAPD (Random amplified polymorphic DNA)
AFLP (amplified fragment length polymorphism)
Nested PCR
Bio PCR
Microrraays
ELISA (Enzyme lined immune sorbent assay)
FISH hybridisation (Fluorescence in situ hybridization )
NASBA (Nucleic acid sequence based amplification)
SAGE (serial analysis gene expression)
Blotting techniques (DNA, RNA and Protein)
DNA barcoding
Insitu hybridization
RNA interference
NO FOOD, NO LIFE
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)