Escolar Documentos
Profissional Documentos
Cultura Documentos
(Sweetpotato/Kumara)
Post-Entry Quarantine
Testing Manual
November 2012
www.mpi.govt.nz
SCOPE ....................................................................................................................................................... 1
INTRODUCTION ..................................................................................................................................... 1
IMPORT REQUIREMENTS................................................................................................................... 3
PESTS ........................................................................................................................................................ 3
4.1
4.2
Regulated pests for which specific tests are required ................................................................... 4
5.
PROPAGATION, CARE AND MAINTENANCE IN POST-ENTRY QUARANTINE .................... 4
6.
7.
5.1
Whole plants..................................................................................................................................... 4
5.2
5.3
Pollen ................................................................................................................................................ 5
INSPECTION ............................................................................................................................................ 5
TESTING ................................................................................................................................................... 6
7.1
Specific tests for nursery stock ....................................................................................................... 7
7.1.1
Graft inoculation ......................................................................................................................... 8
7.1.2
Herbaceous indexing................................................................................................................. 10
7.1.3
Serological and molecular assays ............................................................................................ 11
7.1.3.1
Enzyme-linked immunosorbent assay (ELISA) .......................................................... 11
7.1.3.2
Polymerase chain reaction (PCR)................................................................................. 12
7.1.3.2.1 Virus reverse transcription-PCR .............................................................................. 14
7.1.3.2.1.1 Sweet potato chlorotic stunt virus ........................................................................ 18
7.1.3.2.1.2 Sweetpotato leaf curl virus ................................................................................... 18
7.1.3.2.1.3 Sweetpotato mild speckling virus ......................................................................... 18
7.1.3.2.1.4 Sweetpotato vein mosaic virus .............................................................................. 18
7.1.3.2.1.5 Tobacco streak virus ............................................................................................. 18
7.1.3.2.2 Phytoplasma PCR ....................................................................................................... 19
7.1.3.2.2.2 Sweetpotato little leaf phytoplasma ................................................................... 21
7.1.3.2.3 Bacteria PCR .............................................................................................................. 21
7.1.3.2.3.1 Dickeya chrysanthemi .......................................................................................... 21
7.1.4
Bacterial isolation on media ..................................................................................................... 22
7.1.4.1
Dickeya chrysanthemi (basonym. Erwinia chrysanthemi) ........................................... 22
7.1.5
Microscopic inspection for mites ............................................................................................. 23
7.1.5.1
Tetranychus evansi ......................................................................................................... 23
8.
CONTACT POINT ................................................................................................................................. 24
9.
ACKNOWLEDGEMENTS .................................................................................................................... 24
10. REFERENCES ........................................................................................................................................ 24
Appendix 1. Symptoms of significant regulated pests of Ipomoea batatas .................................................. 27
1.1
1.2
1.3
1.4
1.5
1.6
ii
1.7
1.8
1.9
1.10
1.11
Sweetpotato little leaf phytoplasma ............................................................................................. 29
Appendix 2. Virus symptoms on graft inoculated Ipomoea setosa ............................................................... 30
2.1
2.2
2.3
2.4
2.5
2.6
Sweetpotato leaf curl virus + Sweetpotato feathery mottle virus ................................................... 31
Appendix 3. Protocols referenced in manual ................................................................................................. 32
3.1
3.2
iii
1.
SCOPE
The scope of this manual is limited to Ipomoea batatas and Ipomoea setosa nursery stock
(whole plants and plants in tissue culture), seed for sowing and pollen of Ipomoea species
permitted entry into New Zealand as listed in the Ministry for Primary Industries (MPI)
Plants Biosecurity Index (http://www.maf.govt.nz/cgi-bin/bioindex/bioindex.pl). At the date
of publication of this manual, these species were as follows:
Ipomoea alba
Ipomoea aquatica
Ipomoea arborescens
Ipomoea batatas
Ipomoea brasiliensis
Ipomoea cairica
Ipomoea carnea
Ipomoea horsfalliae
Ipomoea imperialis
Ipomoea lobata
Ipomoea minuta
Ipomoea nil
Ipomoea noctiflora
Ipomoea palmata (syn. Ipomoea cairica)
Ipomoea pes-caprae
Ipomoea platensis
Ipomoea purpurea
Ipomoea quamoclit
Ipomoea sepacuitensis
Ipomoea setosa
Ipomoea sloteri
Ipomoea tricolor
Ipomoea tuberosa (syn. Merremia
tuberosa)
2. INTRODUCTION
Sweetpotato (Ipomoea batatas (L.) Lam.), a member of the family Convolvulaceae, probably
originated in Central or South America, where it has been a food source for over 55,000
years. Sweetpotato was taken to Spain and early Spanish explorers are believed to have taken
it to the Philippines and East Indies; from there it was soon carried to India, China, and
Malaysia by Portuguese voyagers. It is not fully known how sweetpotato arrived in
Polynesia, but it has been used on many of the islands in the South Pacific Ocean for at least
2000 years (Clark & Moyer, 1988).
Sweetpotato is grown in a wide range of environments under a range of farming systems,
from the humid tropics to mild temperate zones, and from sea level to 2700 m altitude.
Annual global production of sweetpotato currently exceeds 124 million tonnes. More than
95% of the global sweetpotato crop is grown in developing countries. China is the world's
largest producer, accounting for more than 90%. Vietnam, Indonesia, and Uganda all grow
more than two million tonnes per year. India and Rwanda each harvest more than a million
tonnes annually. Of the 82 developing countries where sweetpotatoes grow, 36 are in Africa,
22 in Asia, and 24 in Latin America. Around 40 countries count sweetpotato among the five
most important food crops produced on an annual basis.
The per capita income provided by sweetpotato is one of the lowest among the major food
crops. Its potential benefit to poor farm households and urban consumers is only now being
considered. Sweetpotato actually produces more edible energy per hectare per day than any
other major food crop.
Sweetpotato is a perennial plant cultivated as an annual crop and propagated vegetatively.
The sweetpotato plant is a prostrate vine system that expands horizontally and develops a
shallow canopy. The sweetpotato plant produces several different types of thick and thin
roots. Thick roots can differentiate into either pencil roots or storage roots, the latter
being used for human consumption. Sweetpotatoes are not tubers as they are initiated at the
first stem node below the soil line, to which they are attached by a stalk of thinner root (Clark
& Moyer, 1988).
Although known for its tolerance to drought and its sensitivity to saturated soil condition,
sweetpotato requires sufficient water and nutrients to produce good yield. Non-rooted stem
cuttings (20-40 cm) with 5-8 nodes are harvested from storage roots laid out in nursery beds,
and transplanted into the field. Sweetpotato can be cultivated continuously throughout the
year in tropical regions, but in temperate regions the crop is planted in spring when the risk of
frost is reduced. The crop requires a minimum frost-free period of 120-150 days and average
daily temperatures of 22-24C along with good rainfall and good drainage (Clark & Moyer,
1988).
The flesh of sweetpotato can be white, purple, orange or yellow. Yellow and orange-fleshed
varieties are valuable for their carotene (provitamin A) content. Skin colour ranges from
nearly white through shades of buff to brown, or through pink to copper, even magenta and
purple.
In New Zealand, sweetpotato (known as kumara) is a crop of cultural importance and an
important food source. Kumara was introduced by Maori when they settled in New Zealand
from Polynesia. Cultivation of this crop was undertaken on a large scale because of its
importance as a food source. With the arrival of European settlers, other carbohydrate crops
such as potato, wheat, and corn displaced sweetpotato in dietary importance but not the
crops place in Maori culture. Since the 1950s, after efforts to select improved clones,
production has steadily increased along with consumption as people rediscover kumara.
The local cultivar Owairaka Red, released in 1954, comprises 80% of the crop. Other
cultivars include Toka Toka Gold, selected in 1972 (14%) and Beauregard (introduced in
1993). A range of other local varieties are also grown, usually in garden plots.
The area around Dargaville in the Kaipara district produces 85% of the national crop.
Smaller plantings of approximately 5% are found around Auckland and Bay of Plenty. The
area planted annually is approximately 1,100 hectares producing about 26,500 tonnes, with
yields averaging 20 t/ha. Plantings range in area from garden plots to 30 ha, averaging 10 ha.
Most of New Zealands production is for local fresh consumption although increasing
amounts are processed and/or exported. A thorough review of this crop and its place in New
Zealand agriculture is presented by Lewthwaite (1997).
3. IMPORT REQUIREMENTS
The import requirements for I. batatas and I. setosa nursery stock (whole plant and plants in
tissue culture) are set out in MPIs import health standard Importation of Nursery Stock
(http://www.biosecurity.govt.nz/files/ihs/155-02-06.pdf). Imported nursery stock must meet
the general requirements (sections 1-2) and the specific requirements detailed in the
Ipomoea batatas schedule. On arrival in New Zealand, the nursery stock must be grown
for a minimum period of 3 months in a Level 3 post-entry quarantine facility where it will be
inspected, treated and/or tested for regulated pests.
The import requirements for Ipomoea seed for sowing are set out in MPIs import health
standard Importation of Seed for Sowing (http://www.biosecurity.govt.nz/files/ihs/155-0205.pdf). Imported seed is only required to meet the general requirements (sections 1-2) and
there are no specific requirements for the genus. An import permit is not required and seed
meeting the import requirements is given biosecurity clearance at the border without the need
for post-entry quarantine.
The import requirements for pollen are stated in section 2.2.3 in MPIs import health standard
Importation of Nursery Stock (http://www.biosecurity.govt.nz/files/ihs/155-02-06.pdf ) and
further details can be found in section 5.3 of this manual.
4. PESTS
The following section lists regulated pests of I. batatas and I. setosa nursery stock that
require generic or specific measures.
4.1 Regulated pests for which generic measures are required
Insects:
Cylas formicarius
Cylas puncticollis
Euscepes postfasciatus
Nematodes:
Meliodogyne incognita
Rotylenchulus reniformis
Pratylenchus coffeae
Pratylenchus brachyurus
[Fig. 1.1]
[Fig. 1.2]
Fungi:
Elsino batatas
Helicobasidium mompa
[Fig. 1.6]
Bacteria:
Pseudomonas batatas
Streptomyces ipomoea
Xanthomonas batatae
Xylella fastidiosa
[Fig. 1.5]
Viruses:
Sweetpotato chlorotic fleck virus
Sweetpotato latent virus
Sweetpotato ringspot virus
Sweetpotato virus C6
[Fig. 1.8]
Bacteria:
Dickeya chrysanthemi
[Fig. 1.7]
Phytoplasma:
Sweetpotato little leaf phytoplasma
[Fig. 1.11]
Viruses:
Sweetpotato caulimo-like virus
Sweetpotato chlorotic stunt virus
Sweetpotato leaf curl virus
Sweetpotato leaf speckling virus
Sweetpotato mild speckling virus
Sweetpotato vein mosaic virus
Sweetpotato yellow dwarf virus
Tobacco streak virus
5.
Whole plants should be planted into sufficiently sized pots (eg 3 L minimum) containing
50:50 (v/v) pasteurised peat:pumice planting media and a few grams of slow-release fertiliser
with trace elements (e.g. Osmocote). Nodal cuttings should be taken from growing vines to
maintain the clone and facilitate quarantine examination and testing. Cuttings with one or
two leaves and at least two nodes can be rooted directly in pasteurised pumice sand or perlite
before transferring to planting media. It would be worth preserving clonal material in tissue
culture as a back-up resource, and to preserve any established virus-free status.
Plants in tissue culture
Tissue culture plantlets can be sub-cultured after arrival by cutting into nodal sections and
placing into new tissue culture vessels with fresh nutrient media (e.g. Murashige and Skoog
media).
Plantlets to be tested are carefully excised from the tissue culture vessel and washed to
remove any remaining agar and planted into pots of planting media containing 50:50 (v/v)
pasturised peat:perlite or 50:50 (v/v) peat:vermiculite. The plantlets must be protected from
desiccation for approximately three weeks by covering initially with a vented plastic tub or
bag. Alternatively, the plants can be misted regularly to keep the planting media moist, and
to maintain a high relative humidity. Pots should be placed in bright light, but not direct
sunlight during the three weeks. After this period, any coverings should be removed and the
plants moved to higher light intensity.
5.3 Pollen
Anthers can be collected from mature but unopened flowers and dried in warm, light
conditions. Following this drying period, pollen should be collected into a centrifuge vial or
into gel capsules and stored at 4C in a sealed container in the presence of a strong desiccant
such as calcium chloride.
6. INSPECTION
The inspection requirements for the operator of the facility are set out in the MPI
Biosecurity Authority Standard PBC-NZ-TRA-PQCON
(http://www.biosecurity.govt.nz/files/regs/stds/pbc-nz-tra-pqcon.pdf )
Photographs of symptoms caused by significant regulated diseases can be found in Appendix
1. However, please note that pot-grown sweetpotato plants can be prone to nutrient
deficiencies if not adequately fertilised and nutrient deficiencies can resemble virus infection,
e.g. chlorosis and necrosis. Symptoms related to nutrient deficiencies can be found in
Appendix 2. Further information on nutrient deficiencies is described in Clark & Moyer
(1988).
7.
TESTING
Each of the specific tests required in the import health standard (as described in section 4 and
summarised in Table 1) must be done irrespective of whether plants exhibit symptoms. This
testing is required to detect latent infections.
Samples should be tested as soon as possible after removal from the plant. If samples have to
be stored before testing, the plant material must be kept whole, all surface water must be
removed, and the material stored in a plastic bag at 4oC. Samples that become partially
decayed or mouldy must not be tested, and further samples should be collected.
Inspection for mites
Inspection for mites is performed once whole plants or tissue culture plants have established
successfully in planting media and have produced stems with at least 10-15 nodes.
Inspection should take place before samples are taken for other testing methods. Using a
hand lens, the underside of all leaves must be inspected for mite eggs, nymphs, adults and
symptoms of mite presence. Following this, the 3 youngest leaves of each plant, plus any
suspect leaves showing the presence of mites must be collected for further examination under
a binocular microscope. See section 7.1.5 for further details.
Indexing tests
Graft inoculation: Grafting can begin when the whole plants or tissue culture plants have
established successfully in planting media and have produced stems with at least 10-15
nodes. Grafting should take place before leaf samples are collected for other testing methods.
See section 7.1.1 for further details. Each plant in the glasshouse must be tested individually
by graft indexing
Herbaceous indexing: Virus testing should be done in spring (or under spring-like
conditions) when new growth has occurred. At least two fully expanded leaves must be
sampled from each of two different branches of the main stem, one a younger leaf and one an
older leaf from a mid-way position. Each plant in the glasshouse must be tested individually
by herbaceous indexing. See section 7.1.2 for further details
PCR and ELISA testing
Viruses: For virus-testing of I. batatas by PCR and ELISA, it is recommended to test the
graft-inoculated indicator plants rather than the original test plants. However, original I.
setosa plants can be tested directly. Virus testing should be done in spring (or under springlike conditions) when new growth has occurred. At least two fully expanded leaves must be
sampled from each of two different branches of the main stem, one a younger leaf and one an
older leaf from a mid-way position. The sampled leaves from each plant must be bulked
together and tested as soon as possible after removal from the host. See section 7.1.3 for
further details.
Bacteria and phytoplasma: Bacteria and phytoplasma testing must be carried out using the
original I. batatas and I. setosa plants and should be done in summer (or under summer-like
conditions). For each plant, at least two fully expanded leaves must be sampled from each of
two different branches of the main stem, one a younger leaf and one an older leaf from a midway position. Detection of both bacteria and phytoplasma requires testing of leaf petioles
and mid-veins. The sampled leaves from each plant must be bulked together and tested as
soon as possible after removal from the host. See section 7.1.3 for further details
Table 1: Summary of the regulated pests for I. batatas and I. setosa indicating the
specific tests that are required (), alternative () or optional ()
Organism Type
Graft
Inoculation1
Mites
Tetranychus evansi
Bacterium
Dickeya chrysanthemi
Phytoplasma
Sweetpotato little leaf
phytoplasma
Viruses
Sweetpotato caulimo-like virus
Sweetpotato chlorotic stunt
virus
Sweetpotato leaf curl virus2
Sweetpotato leaf speckling
virus
Sweetpotato mild speckling
virus
Sweetpotato vein mosaic virus
Sweetpotato yellow dwarf virus
Tobacco streak virus
1
Herbaceous
Indexing
ELISA
PCR
Isolation
on media
Inspection
7.1
Each plant must be tested separately with the following exceptions, samples from up to 5
plants may be bulked for testing provided that either:
(a) the plants are derived from a single imported plant or plant established from a storage
root from which separate cuttings have been taken upon arrival in New Zealand, in
the presence of a MPI inspector; or
(b) in the case of tissue culture where plants are clonal, and this is confirmed by evidence
from the national plant protection organisation in the exporting country.
7.1.1
Graft inoculation
Each I. batatas plant must be tested by graft inoculation using a minimum of 3 replicate
indicator plants of either I. setosa or I. nil Scarlet O Hara. Indicator plants must be
maintained in a healthy, vigorous state, as symptoms associated with abiotic stresses, such as
water and nutrient deficiencies, may mask and interfere with observations of disease
symptoms. The indicator plants can be grown from seed or from young cuttings. If using
seed, sow 3-4 weeks before grafting.
Sweetpotato seed requires scarification prior to germination. Indicator seeds are soaked in
concentrated sulphuric acid (98%) for 20 minutes (I. nil) or 60 minutes (I. batatas). Seeds
are then rinsed in running tap water 3-4 times prior to planting in moist planting media.
The method for propagating sweetpotato plants from seed is described in full by Saladaga et
al. (1991).
The indicator plants are ready for grafting when they have two or more fully expanded
leaves.
To avoid cross-contamination of plants during the grafting process, use a sterile scalpel for
each sweetpotato plant to be tested.
Recommended method
1. Begin grafting by cutting indicator plants back to 2 true leaves.
2. Sweetpotato plants are tested by wedge-grafting. Each sweetpotato plant that is to be
used for indexing should be established with a minimum of five nodes. Remove a branch
from the sweetpotato plant to be indexed and cut the branch into 5 sections, each
containing a node with a fully expanded leaf attached.
3. Wedge-graft each node section onto a separate indicator plant.
4. To prevent desiccation, wrap the graft with parafilm, or similar.
5. Cover the whole plant with a plastic bag to reduce airflow around the graft.
6. Remove the plastic bag 5-7 days after grafting.
7. Fertilise the indicator plant with a slow-release fertiliser (e.g. Osmocote) and insert a
bamboo-stake into the pot to support the growth of the plant.
8. Grow the indicator plants to at least 10-15 nodes; this will take approximately 3-5 weeks.
During the growth period, monitor the indicator plants daily for virus symptoms which
may only show for a short period of time.
9. Some sweetpotato grafts may grow faster than the indicator plant, cut any sweetpotato
growth back to ensure the indicator plant grows well.
10. At the end of the 3-5 week growth period, cut the indicator plants back to 1-2 buds and
re-grow for 3-5 weeks. Re-growth should again be closely monitored daily for virus
symptoms.
11. A positive control must be included with each batch of inoculations. For the positive
control, graft a sweetpotato plant known to be infected with a non-regulated virus, e.g.
Sweetpotato feathery mottle virus (SPFMV).
12. It is recommended to include a negative control with each batch of inoculations. For the
negative indicator, cut back to 2 true leaves, as for grafted plants, but do not graft.
Note: Sweetpotato plants are sensitive to some pesticides and spray damage can induce
mosaic-like symptoms. In addition, plants suffering from nutrient deficiencies can show
leaf chlorosis and necrosis.
Interpretation of results
Symptoms on I. setosa usually appear within 2-4 weeks, and on I. nil around one week.
However, the severity of virus symptoms and length of time before they appear on the
indicator plants depends upon the virus and the amount of virus inoculum present in the
scion. The graft inoculation results will only be considered valid if:
(a) no symptoms are produced on the negative control (non-grafted) indicator plant; and
(b) the expected symptoms are produced on the indicator hosts with the positive control
(non-regulated virus). If SPFMV was used as the positive control, the following
symptoms will be produced on the indicator plants:
I. setosa vein clearing followed by remission.
I. nil systemic vein clearing, vein banding, ringspots.
The symptoms produced by each of the regulated viruses on the indicator species I. setosa
and I. nil are described below.
Sweetpotato caulimo-like virus:
I. setosa chlorotic flecks along the secondary veins and interveinal chlorotic spots on
leaves.
Sweetpotato chlorotic stunt virus:
I. setosa stunting, yellowing and leaf deformation, although symptoms maybe mild
depending on isolate.
I. nil stunting, yellowing and leaf deformation, although symptoms maybe mild
depending on isolate.
Sweetpotato leaf curl virus:
I. setosa curling of young leaves.
I. nil curling of young leaves.
Sweetpotato leaf speckling virus:
I. setosa chlorotic and necrotic spotting, dwarfing and leaf curling.
I. nil chlorotic and necrotic spotting, dwarfing and leaf curling.
Sweetpotato mild speckling virus:
I. setosa mild mosaic sometimes observed in first two true leaves.
Sweetpotato vein mosaic virus:
I. setosa systemic vein-clearing and mosaic.
I. nil systemic vein-clearing and mosaic.
Sweetpotato yellow dwarf virus:
I. setosa chlorotic leaf mottling.
7.1.2
Herbaceous indexing
Each I. batatas and I. setosa plant must be tested for mechanically-transmitted regulated
viruses using herbaceous indicators, this is in addition to graft inoculation. Sap must be
inoculated onto two plants of each herbaceous species as follows: Chenopodium quinoa,
Nicotiana benthamiana, N. clevelandii and N. tabacum.
It is important that the pre- and post-inoculation growing conditions of the herbaceous
indicator plants promote their susceptibility. Plants must be grown at 18-25oC. The stage of
development to ideally inoculate the indicator plants is 4-6 fully expanded true leaves for
Chenopodium spp., and 4 fully expanded leaves for Nicotiana spp.
Recommended method
1. Place indicator plants in dark for 16-24 hours prior to inoculation to increase
susceptibility.
2. Grind leaf tissue (approximately 1/4; w/v) in 0.1 M sodium phosphate buffer (pH 7.5),
containing 5% (w/v) polyvinylpyrrolidone (PVP-40) and 0.12% (w/v) sodium sulphite
(Na2SO3). A negative (inoculation buffer only) and a positive control must be included in
each batch of inoculations. The positive control is a non-regulated virus which is
moderately transmissible and produces clear symptoms on the herbaceous indicators, (e.g.
Arabis mosaic virus). The plants must be inoculated in the following order:
(a) inoculation buffer only; then
(b) imported plants to be tested; then
(c) positive control (non-regulated virus).
3. Select two young fully expanded leaves preferably opposite leaves, to be inoculated on
each plant and mark them by piercing holes with a pipette tip.
4. Lightly dust the leaves with Celite or carborundum powder. Alternatively, a small
amount of Celite or carborundum powder may be mixed with the sap extract.
5. Using a gloved finger gently apply the sap to the marked leaves of the indicator plants,
stroking from the petiole towards the leaf tip while supporting the leaf below with the
other hand.
6. After 3-5 minutes rinse inoculated leaves with water.
7. Grow inoculated plants for a minimum of 4 weeks. Inspect and record plants twice per
week for symptoms of virus infection.
The Arabis mosaic virus positive control may be obtained from:
1. ATCC Cat. No. PV-192, PV-589, PV-590 (http://www.atcc.org).
2. DSMZ Cat. No. PV-0045, PV-0046, PV-0215, PV-0216, PV-0217, PV-0230, PV-0232
(http://www.dsmz.de).
3. The MPI (see the Contact Point, section 8) (available as freeze-dried leaf material or
nucleic acid). A charge may be imposed to recover costs.
Interpretation of results
The herbaceous indexing results will only be considered valid if:
(a) no symptoms are produced on the indicator hosts with the negative control
(inoculation buffer only); and
(b) the correct symptoms are produced on the indicator hosts with the positive control
(non-regulated virus). If Arabis mosaic virus was used as the positive control, the
following symptoms will be produced on the herbaceous indicators:
C. quinoa local lesions, and systemic chlorotic mottling.
10
7.1.3
11
Antisera
Positive/negative
control2
Agdia Cat No.
LNC 27200
Agdia Cat No.
LNP 27200
Agdia Cat No.
LPC25500
Catalogue numbers for the complete reagent sets are given, the antisera and reagents can
also be purchased separately.
2
The positive control is included if the Pathoscreen set is purchased.
Further information about the kits and the supplier listed in Table 2 can be found at the
following website:
Agdia Incorporated, USA (http://www.agdia.com).
Interpretation of results
A result is considered positive if the mean absorbance of the two replicate wells is greater
than 2 times the mean absorbance of the negative control. The test will only be considered
valid if:
(a) the absorbance for the positive and negative controls are within the acceptable range
specified by the manufacturer; and
(b) the coefficient of variation (standard deviation / mean 100), between the duplicate
wells is less than 20%.
If the test is invalid, it must be repeated with freshly-extracted sample. Samples that are close
to the cut-off must be retested or tested using an alternative method recommended in the
import health standard (see Table 1).
12
Table 3: PCR primers used for the detection of regulated pests of I. batatas and I. setosa,
and plant internal controls
Target
organism
Bacterium
Dickeya chrysanthemi
(Use both assays to
detect all pathovars)
Primer
name
Sequence (5-3)
ADE1
ADE2
GATCAGAAAGCCCGCAGCCAGAT
CTGTGGCCGATCAGGATGGTTTTGT
CGTGC
GGTAAAGGGTCTATCATGCG
CCTTCACCATACATAATTTGGA
72
420
Nassar et al.,
1996
47
760
Waleron et al.,
2002
AAGAGTTTGATCCTGGCTCAGGATT
53
recAF
recAR
Phytoplasma
Sweetpotato little leaf P1
phytoplasma
P7
R16F2
R16R2
Phyto-F
Sweetpotato mild
speckling virus and
Sweetpotato vein
mosaic virus
Tobacco streak virus
Internal Control
Plant DNA control
50
60
75
Christensen et
al., 2004
CGAATCAACGGATCGGAATT
CCACCGACTATTACATCACCACTCT
(MGB)FAM-ATCCCAACGTGTTTATCT
A-NFQ3
EASPCSV-38F GGAGTTTATTCCCACCTGTYTATCT
EASPCSV-126R GTAATTGCGAAGAATCYAAAACCT
EASPCSV-67P2 FAM-CGGCTACAGGCGACGTGGTTG
TTG-NFQ3
SPG1
ATCCVAAYWTYCAGGGAGCTAA
SPG2
CCCCKGTGCGWRAATCCAT
SPLCV-F
GGCGCCTAAGTATGGCTGAA
SPLCV-R
AACCGTATAAAGTATCTGGGAGT
GGT
(MGB)FAM-GTGGGACCCTTTGCSPLCV-P2
NFQ3
Oligo1n
ATGGTHTGGTGYATHGARAAYGG
Oligo2n
TGCTGCKGCYTTCATYTG
60
71
Kokkinos &
Clark, 2006
60
90
N. Boonham
(Unpublished)
58
934
Li et al., 2004
66
60
Kokkinos &
Clark, 2006
50
327
Marie-Jeanne et
al., 2000
48
300
Untiveros et al.,
2010
SPCSV-F
SPCSV-R
SPCSV-P2
IlarlF5
IlarlR7
GCNGGWTGYGGDAARWCNAC
AMDGGWAYYTGYTYNGTRTCACC
Gd1
Berg54
ACGGAGAGTTTGATCCTG
50-62 1500 Andersen et al.,
AAAGGAGGTGATCCAGCCGCACCTT
1998
C
GATGCTTCTTGGGGCTTCTTGTT
50-60 180 Menzel et al.,
CTCCAGTCACCAACATTGGCATAA
2002
CGTCGCATTCCAGATTATCCA
60
74 Weller et al.,
CAACTACGGATATATAAGAGCCAA
2000
AACTG
FAM-TGCTTACGCTGGATGGAATG
CCCT- NFQ3
Nad5-s
Nad5-as
COX-F
COX-R
COX- P2
CGTCCTTCATCGGCTCTT
Band Reference
(bp)
ACGACTGCTAAGACTGG
TGACGGGCGGTGTGTACAAACCCCG
CGTACGCAAGTATGAAACTTAAAG
GA
TCTTCGAATTAAACAACATGATCCA
FAM-TGACGGGACTCCGCACAAGCG
-NFQ3
Phyto-R
Phyto-P2
Viruses
Sweetpotato chlorotic
stunt virus
(Use both assays to
detect East & West
African strains)
TM
(C)
13
7.1.3.2.1
14
*Li et al. (2004) PCR only, for all other primers, adjust water volume accordingly
Temperature
50oC
94oC
94oC
See Table 3
Elongation
72oC
Final elongation
72oC
Time
30 min
2 min
30 sec
30 sec
30 to 45 sec (virus/bacteria)
1 min (phytoplasma)
7 min
No. of cycles
1
1
40
1
15
16
Temperature
50C
95oC
95oC
See Table 3
Time
30 min
2 min
10 sec
40 sec
No. of cycles
1
1
40
Temperature
50C
Time
2 min
95C
Denaturation
Annealing & elongation
95C
See Table 3
2 min (Invitrogen)
5 min (Roche)
10 sec
40 sec
No. of cycles
1
1
40
17
Positive control material, in the form of nucleic acid, for Sweetpotato chlorotic stunt virus
and Sweetpotato leaf curl virus and Tobacco streak virus may be obtained from MPI (see the
Contact Point, section 8). Positive control material for Sweetpotato vein mosaic virus and
Sweetpotato mild speckling virus is currently unobtainable; however, an alternative Potyvirus
may be used for the PCR. Potyvirus positive controls in the form of nucleic acid may also be
obtained from the MPI. A charge may be imposed to recover costs.
7.1.3.2.1.1 Sweet potato chlorotic stunt virus
Plants must be tested for Sweetpotato chlorotic stunt virus by real-time PCR using the primer
pairs listed in Table 3. See section 7.1.3.2.1 for details of test methods and interpretation of
results. Please note that SPCSV should be tested with both sets of primers listed in Table 3 in
order to detect both East and West African strains.
7.1.3.2.1.2 Sweetpotato leaf curl virus
Plants must be tested for Sweetpotato leaf curl virus by PCR or real-time PCR using the
primer pairs listed in Table 3. See section 7.1.3.2.1 for details of test methods and
interpretation of results. Please note the Li et al., (2004) PCR should be cycled as shown in
Table 11
Table 11: Cycling conditions for SPLCV PCR
Step
Initial denaturation
Denaturation
Annealing
Elongation
Final elongation
Temperature
94oC
94oC
58C
68oC
68oC
Time
2 min
30 sec
30 sec
90 sec
3 min
No. of Cycles
1
40
1
18
7.1.3.2.2
Phytoplasma PCR
19
3.
4.
5.
6.
The reaction and cycling conditions can be changed depending on the real-time reagents
and machine used, but this would require validation.
Optional: Perform PCR on the nucleic acid using the COX internal control primers
(Table 3), and using the components and concentrations listed in Table 12 and cycle
under the conditions listed in Table 10.
The following controls must be included for each set of reactions:
(a) Positive control: total DNA or a cloned fragment from the appropriate organism
may be used. If the internal control primers are not used, then the DNA must be
mixed with healthy Ipomoea DNA to rule out the presence of PCR inhibitors; and
(b) no template control: water is added instead of DNA template
When setting up the test initially, it is advised that a negative control (DNA extracted
from healthy Ipomoea leaf tissue) is included.
Analyse real-time amplification data according to the real-time thermocycler
manufacturers instructions.
Table 12: Real-time PCR reaction components for phytoplasma using
Roche LightCycler 480 Probes Mastermix
Reagent
Nuclease-free water
2 Reaction Mix (Roche 04707494001)
10 g/l Bovine Serum Albumin (BSA) (Sigma A7888)
5 M Forward primer (300 nM)
5 M Reverse primer (300 nM)
5 M Dual-labelled fluorogenic probe (100 nM)
DNA
Total volume
20
21
(a) Primers recAF/recAR (Waleron et al., 2002) detects bacteria at the generic level belonging
to the former Erwinia genus; however, sequencing of the resulting recA PCR product will
provide resolution to the sub-species level.
(b) Primers ADE1/ADE2 (Nassar et al., 1996) will detect pectinolytic strains of Dickeya
spp.
Temperature
94oC
94oC
47C
72oC
72oC
Time
3 min
1 min
1 min
2 min
5 min
No. of cycles
1
35
1
7.1.4
Temperature
94oC
94oC
72C
72oC
72oC
Time
3 min
1 min
1 min
2 min
5 min
No. of cycles
1
25
1
Isolation of regulated bacteria from plants is a required test on the Ipomoea IHS. Plants
should be tested separately. Aseptic techniques should be used throughout the test procedure.
22
and the average number of peritichous flagellae is 8-11. On PDA media, depending on the
moisture content, young colonies can be circular, convex, smooth and entire, or sculptured
with irregular margins. After 4-5 days, both types of colonies resemble a fried egg, with a
pinkish, round, raised centre and a lobed periphery, which later becomes feathery.
7.1.5
Microscopic examination of plants for regulated mites is a required test on the Ipomoea IHS.
23
8.
CONTACT POINT
9.
ACKNOWLEDGEMENTS
We would like to acknowledge the following people who contributed to the preparation of
this manual:
Mr John Fletcher (The New Zealand Institute for Plant & Food Research Ltd, Lincoln,
New Zealand) for drafting the introduction and propagation sections of the manual, for
valuable discussion and advice on sweetpotato viruses, and for providing photographs of
virus-infected sweetpotato.
Dr Chris Clark and Ms Mary Hoy (Louisiana State University, USA) for valuable
discussion on sweetpotato viruses, for supplying isolates of SPV2 and SPLCV, and for
supplying several photographs of virus-infected I. batatas and I. setosa.
Dr Steve Lewthwaite (The New Zealand Institute for Plant & Food Research Ltd,
Pukekohe, New Zealand) for providing the front cover image of the sweetpotato cultivar
'Radical' (the first New Zealand sweetpotato cultivar to receive plant variety rights) and
for providing information on seed propagation.
Dr Segundo Fuentes (International Potato Centre (CIP), Peru) for valuable discussion on
sweetpotato viruses.
Ms Susan Sim (Foundation Plant Services, University of California, Davis, USA) for
valuable advice on graft inoculation.
Dr Karen Gibb (Charles Darwin University, Australia) for supplying phytoplasma DNA
and the photograph of the Sweetpotato little leaf phytoplasma.
The American Phytopathological Society (APS) for permission to use images from the
Diseases of Root and Tuber Crops CD-Rom, 2000, St Paul, MN, USA.
10.
REFERENCES
24
25
26
(a)
(a) Galls and egg masses produced by M. incognita on fibrous roots; (b) cracking of fleshy storage roots
associated with injury by M. incognita. (Courtesy W.J. Martin (a) & G.W. Lawrence (b) reproduced with
permission from the Diseases of Root and Tuber Crops CD-ROM, 2002, APS, St Paul, MN, USA).
1.2
Rotylenchulus reniformis
Cracking of fleshy storage roots associated with injury by R. reniformis (Courtesy C.A. Clark reproduced with
permission from the Diseases of Root and Tuber Crops CD-ROM, 2002, APS, St Paul, MN, USA).
27
(b)
(a) Soil pox lesions caused by S. ipomoea on storage roots of I. batatas clone L4-89 (b) I. batatas rootlet rot on
sweetpotato fibrous roots, caused by S. ipomoea. (Courtesy W.J. Martin (a) & (b) reproduced with permission
from the Diseases of Root and Tuber Crops CD-ROM, 2002, APS, St Paul, MN, USA).
28
Leaf symptoms on I. batatas Toka Toka Gold infected with Sweetpotato feathery mottle virus, Sweetpotato
chlorotic fleck virus, and Sweetpotato virus C6. (Courtesy J. Fletcher, The New Zealand Institute for Plant &
Food Research Ltd, Lincoln, New Zealand).
1.10
1.11
29
30
31
Ipomoea Post-Entry Quarantine Testing Manual November 2012
3.2
Phytoplasma DNA enrichment CTAB extraction protocol (Kirkpatrick et al., 1987 and
modified by Ahrens & Seemller, 1992)
1. Grind approximately 0.3 g tissue (petioles, veins) in 3 ml ice-cold isolation buffer (0.1 M
Na2HPO4, 0.03 M NaH2PO4, 10 mM EDTA (pH 8.0), 10% (w/v) sucrose, 2% (w/v) PVP-40;
Adjust pH to 7.6 and filter sterilise. Just prior to use add 0.15% (w/v) Bovine Serum
Albumin (BSA) and 1 mM ascorbic acid).
2. Transfer crude sap to a cold 2 ml micro-centrifuge tube.
3. Centrifuge at 4C for 5 min at 4500 rpm.
4. Transfer supernatant into a clean 2 ml micro-centrifuge tube.
5. Centrifuge at 4C for 15 min at 13000 rpm.
6. Discard the supernatant.
7. Resuspend the pellet in 750 l of hot (55 C) CTAB buffer (2% (w/v) CTAB, 100 mM TrisHCl [pH 8.0], 20 mM EDTA [pH 8.0], 1.4 M NaCl, 1% (w/v) PVP-40). The pellet is
easier to resuspend in a smaller volume of CTAB buffer (e.g. 100 l) then the remaining
volume of CTAB buffer is added (e.g. 650 l).
8. Incubate tubes at 55 C for 30 min with intermittent shaking.
9. Cool the tubes on ice for 30 sec.
10. Add 750 l chloroform:octanol (24:1 v/v) and vortex thoroughly.
11. Centrifuge at 4C or at room temperature for 4 min at 13000 rpm.
12. Carefully remove upper aqueous layer into a clean 1.5 ml micro-centrifuge tube.
13. Add 1 volume ice-cold isopropanol and vortex thoroughly.
14. Incubate on ice for 4 min.
15. Centrifuge at 4C or at room temperature for 10 min at 13000 rpm.
16. Discard supernatant.
32
17. Wash DNA pellets with 500 l ice-cold 70% (v/v) ethanol, centrifuge at 4oC or at room
temperature for 10 min at 13000 rpm.
18. Dry DNA pellets in the DNA concentrator or air-dry.
19. Resuspend in 20 l sterile distilled water. Incubating the tubes at 55oC for 10 min can aid
DNA resuspension.
20. Store DNA at -20C for short-term storage or -80C for long-term storage.
33
Ipomoea Post-Entry Quarantine Testing Manual November 2012