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Tegniese Nuus • Technical News
potatoes
SOUTH AFRICA
Can a leaf sample from a field
planting be considered as an official
sample in the certification process?
Artikel: Anel Espach, Plantovita
INTRODUCTION
Currently in the Potato
Certification Scheme the
use of tuber samples to
test for the presence of virus and bacterial diseases
are prescribed as part of
the certification process.
Properties of the pathogen
and disease allow the
bacterial tests to commence as soon as the
sample has arrived at the
laboratory. Virus tests on
the other hand can only proceed when the tubers are test-ready.
Being test-ready implies that dormancy has been broken, the
tubers were allowed to
sprout, and sprouts of ALL
tubers in a sample are
of a minimum length of
3-5mm.
Based on research
projects, 28 days proved
to be the time before
which most cultivars are
not test ready and the time at which the virus content is sufficient
to reliably detect using the ELISA-based method.
Leaf samples have been used in potato production as a management tool to allow the grower to predict the virus presence of the
progeny tubers in a planting. Decision making by the grower is
thereby facilitated after test results from leaf samples are received.
The use of leaf samples as a management tool is
based on the assumption that high virus content in
the leaf sample indicates an increased probability
that a high percentage of tubers are also infected
with virus.
Questions arose from growers regarding the possibility to use
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leaf samples instead of
tuber samples for certification of seed potatoes – thus
not only as a management
tool, but treating the results
from leaf samples as official
results in certification of the
represented seed lot.
Aspects that have to be
taken into account when
considering what kind of sample should be used, include, but are
not limited to:
1.
2.
3.
4.
Applicable and influencing properties of viruses in question
(Potato Virus Y - PVY and Potato Leaf roll virus - PLRV)
Historic data collected in the certification and testing scenario
in South Africa
The current testing scenario employed at Potato Laboratory
Services, South Africa
Logistical and practical aspects to be considered
Advantages and disadvantages of using leaves as
sample for virus detection include:
•
•
Tannins and starch can cause false readings in a test assay,
with specific reference to the ELISA-based assays currently
used. The limited amount of tannins and starches in leaf
material is advantageous in the generation of reliable results.
It is not necessary to break dormancy in leaf samples –
samples can thus be tested when submitted. This should save
time in the testing process.
Leaf samples have the disadvantage of not being a true representation of what the virus status of the
tubers are and that the leaves does
not have a shelf life.
Any test method has limitations
and aspects to consider during the
implementation of such a method.
Before any method is implemented
knowledge about the advantages and disadvantages of and the
value to an industry, have to be assessed to enable the adoption
Tegniese Nuus • Technical News
potatoes
SOUTH AFRICA
of a method offering the generation of valuable information.
The purpose of determining/predicting the fraction of tubers with virus content is to allow the client to predict yield
and to produce at optimal potential. To employ a test
method that gives a biased indication of what the fraction
of tubers is with virus content does not give the client the
opportunity to produce at optimal potential.
1 THE VIRUSSES
PLRV
Potato leaf roll virus is
introduced into the vascular tissue of the plant
by feeding aphids. Although it is introduced
into the phloem, PLRV
only infects a small
portion of the available
phloem cells and does
not move effectively in
the mesophyll tissue (movement into the mesophyll tissue is
considered short distance movement). For complete systemic infection it is required that the virus can move locally
over short distances, from cell to cell in the host through
plasmodesmata and then further movement over
longer distances through the phloem. In resistant plants,
the virus mostly occurs in the internal phloem bundles and
cannot move from cell-to-cell without the aid of helper
proteins from other viruses.
This implies that exiting the phloem tissue into mesophyll
cells can only happen when the plants are also infected
with other viruses (Barker H, 1987). Movement of PLRV
through sieve elements (long distance movement) is a
passive process and is strongly dependent on metabolite
fluxes which is for most viruses the case. Derrick and
Barker (1997) showed that movement of virus from point
of infection upwards or downwards could be detected
from 7 days after graft inoculation. The virus resides and
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SOUTH AFRICA
multiplies in the companion cells of the phloem tissue of the plant.
In practice, this implicates that current season infection may take
a while to reach tubers and that the amount of virus particles
does not reach detectable levels quickly. Distribution of the virus
in above ground parts (leaves and stem) does not correlate with
the distribution in below ground parts (tubers).
In mixed infections, synergistic interactions between viruses
can exist that can influence the resistance of potato to other
pathogens as well. PLRV specific resistance can manifest as
further limited movement from the foliage to the tubers – with the
consequence that more virus free tubers are borne from plants
with primary (in-season) infection. It has been reported by Barker
and Harrison (1985) that PLRV occurs in lower concentrations in
leaves of cultivars with resistance. Some cultivars have genetically
controlled resistance to the virus infection, but no cultivar is immune to infection and there are no known sources of major gene
resistance. The proportion of virus-free progeny tubers is greater
in cultivars resistant to infection and accumulation than cultivars
that are susceptible.
Resistance mechanisms and virus
distribution through the plant thus
describe the difference in virus
incidence in leaves compared to
tubers.
With respect to visual observations - visual inspections and
indexing plants by visual inspections are complicated and inconsistent especially at high temperatures due to masking of disease
symptoms in warm conditions. It has been reported by Loebenstein et al (1997) that PLRV is not always detectable in leaves
particularly when plants are grown at temperatures around
30°C and higher. Symptoms of PLRV infection may be absent
in infection that occurred later in the season making sampling of
symptomatic leaves a challenge.
PVY
As mentioned previously, any test method has limitations and
must be considered in implementation of such a method. Disadvantages of using tuber samples can be illustrated in the PVY
testing scenario. PVY is underestimated by using tuber material
as sample during the ELISA test, whether dormant or actively
sprouting. This was shown in a research project conducted at
the University of Aberdeen in Idaho by Jonathan Whitworth et
al in February 2012 and confirmed results obtained by Singh
and Somerville (1986). Siitari and Kurppa (1987) demonstrated
that virus detection in tubers have lower sensitivity in comparison
with leaf samples due to the interference of tannins and starches.
The underestimation was because of distribution of virus through
the plant and the effect on sensitivity of the tuber matrix. To limit
the effect these disadvantages have on the test result, scientists
allow the tubers to sprout or they employ the so-called “growout” method and then after sprouting use as little tuber flesh as
needed to complete the test. The use of fresh leaf material is of
utmost importance since leaf material showing signs of deterioration do contribute to false positive results in test regimes.
A number of potato varieties display latent infection (no or little
symptoms) in the foliage. This makes indexing by visual inspection (as also in the case of PLRV) extremely inconsistent and
rendering it essential to use tuber samples for virus testing.
Singh and Santos-Rojas (1983) compared detection of PVY
primary infection using the ELISA and indicator hosts. In this
study, they found that using the strains and cultivars specific to
the region (Canada), that the ELISA could only detect primary
infection in the leaves from 4 weeks post inoculation. This implies
that very recent infection in leaves would go undetected when
leaf samples are used as sample. Also, PVY symptoms were
more obvious in green-house plants than field grown plants,
emphasizing the effect that environmental conditions have on
pathogenesis. Although it can take no more than a few hours to
reach exponential phase of replication in a cell, it can take days
to weeks to develop a complete systemic infection. As in the case
of PLRV, detection of in season infection remains inconsistent. The
distribution of virus into progeny varies significantly – causing
leaf samples to be an unreliable sample in predicting the virus
content of progeny tubers.
2 HISTORIC DATA COMPARING LEAF AND TUBER
SAMPLES
Leaf samples have been used more regularly as management
tool in the potato industry since 2004. The experience is that
growers in some regions use this tool more frequently than those
in other regions for reasons only speculated about.
The leaf samples are drawn by the growers themselves and
submitted to a laboratory participating in the Potato Laboratory
Services (accredited by ICCSP) group. Since leaf samples are
drawn just before haulm killing, and when the grower sees it fit,
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Tegniese Nuus • Technical News
potatoes
SOUTH AFRICA
arrangements has to be
made with the regional
laboratory when the
sample needs to submitted. It mostly happens
that routine, scheduled
testing has to be re-scheduled to accommodate
leaf samples since the
condition of leaf samples
is crucial. Decayed leaf
samples give rise to false
positive results. The
growers thus determine
time of sampling, growth
stage of the plant, sample
size and representation
from a planting. For some
growers, Potato Certification Service submits seed lists containing
information about the sample/s. Other growers prefer to communicate with the laboratory directly.
Samples are sent to the regional laboratories and stored in a
storage facility that allow temperature and humidity control. The
samples are allowed to sprout in the storage facility to facilitate
sensitive determination of virus presence in tubers. The reasons
for sprouting are based on advantages and disadvantages of
leaf and tuber samples as matrix and include the generation of
sprouts to allow limited tuber flesh into the sample and ensure
that virus titres are at detectable levels. Tubers are used as testing
matrix because as mentioned, detection of virus in leaves
does not compare sufficiently to virus occurrence in
tubers.
Since 2004, samples have been submitted to the individual
laboratories. Generated results have been compared where
information allowed it, to results of tests performed on tuber
samples. In total, data from 741 leaf samples were collected and
compared with tuber samples from different regions. Comparison
of 428 samples was possible. A national average of 30% of the
samples had results that graded seed lots in the same generation.
70% of the samples showed a difference between the
generated leaf and tuber sample test results in terms
of generation graded. The inconsistency in results can be
attributed to several factors:
Although testing is destructive,
the remainder of the tuber samples already submitted to the testing process is stored at the testing facility until the disseminated
results are accepted by the authority and / or grower. If not, the
interested party can call a dispute and the stored sample can be
submitted for the dispute test in the case of PVY. For PLRV, store
samples are drawn.
• Virus properties
• Disease properties
• Sampling inconsistency
4. LOGISTICAL AND PRACTICAL ASPECTS IN CONSIDERATION
3 THE CURRENT SCENARIO AT POTATO LABORATORY
SERVICES
Tuber samples are drawn by Potato Certification Services (PCS)
representing seed lots from plantings registered as seed plantings.
After breaking of the dormancy
of tubers and subsequently sufficient sprouting, the samples are
subjected to the DAS-ELISA assay specific for the pathogen in
question. Generated results are
disseminated from the laboratories to the certification authority
as percentage of tubers infected.
This testing regime allows authorities, growers and clients of
growers to predict the percentage of infected tubers in a seed lot
at the time of harvesting.
At laboratories nationally, roughly 3400 - 400 tuber equivalent
samples are tested each year.
From the beginning of a season, when in some regions the samples grown in that particular region are submitted to the laboratory in the first six weeks of the season, it takes up to four months
and longer to test all submitted samples at optimum capacity.
Although the 28 day waiting period contributes to this time, once
dormancy is broken, samples can only be tested as capacity of
the laboratory allows.
This imply that if 500 samples are submitted to a laboratory in a
season, dormancy is broken and samples is test-ready all at once,
even with five roller presses 500 samples can only be completed
after more than one month. This is however not the situation.
Dormancy cannot be broken all at once due to cultivar differences and different planting dates. Issues like the latter stretches
a testing season for another two months or more. The average
capacity of laboratories is three roller presses to allow sample
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SOUTH AFRICA
processing. Facilities and cost implications do not allow further
expansion. The shelf-life of tubers allows storage and keeping
until testing schedule permits.
When tuber samples are used, tubers can be routinely counted
to monitor sprouting and start testing. Also, tubers have a shelflife that allows scheduled testing as capacity permits. Since the
condition of leaf samples is of utmost importance, testing of fresh
leaf material is a pre-requisite to testing leaf samples. No laboratory has access to sufficient cold room space to maintain leaf
samples for two days in a good enough condition, nor sufficient
roller press facilities to accommodate and allow the testing of leaf
samples as they are submitted in peak season activity. After two
days of cold-storage the leaves start to go down and risk for false
positives increase.
If leaf samples are the chosen matrix, then in season the plantings have to be sampled and the samples sent to the laboratory
immediately to reach the laboratory within 24 hours (maximum).
Packing of sample is important to allow extended keeping of the
condition. After receipt, samples need to be tested immediately.
In the event of peak season sampling activities, laboratories can
receive more than 50 samples per day. This is much more than
the capacity of any laboratory allows.
CONCLUSION
A facility’s capacity is the one thing that can be changed to
accommodate more samples, however, the intrinsic properties of the viruses and the crops they infect cannot be
changed and remain the motivation why tuber samples are used to predict the virus content of a tuber
seed lot and not the leaves. C
REFERENCES
1
2
3
4
5
6
Barker H and BD Harrison (1985). Restricted multiplication
of potato leaf roll virus in resistant potato genotypes. Annals
of Applied Biology, 107:205-212
Derrick PM and H Barker (1997). Short and long distance
spread of potato leaf roll luteovirus: effects of host genes and
transgenes conferring resistance to virus accumulation in
potato. Journal of General Virology, 78:243-251.
Barker H (1987). Invasion of non-phloem tissue in Nicotiana
clevelandii by potato Leaf roll Luteovirus is enhanced in plants
also infected with Potato Y Potyvirus. Journal of General
Virology, 68:1223-1227.
Siitari H and A Kurppa (1987). Time-resolved Fluoroimmuniassay in the detection of Plant viruses. Journal of
General virology, 68:1423-1428.
Singh RP and TH Somerville (1987). Factors affecting the
detection of Potato virus Y in tubers by Enzyme-linked immunosorbent assay (ELISA). Indian Journal of Plant Pathology
4(1): 91-97.
Loebenstein G, Akad F, Filatov V, Sadvakasova G, Manadilova, A, Bakelman H, Teverovsky E, Lachmann O, and David A
(1997). Improved detection of Potato LeafrollLuteovirus in
Leaves and Tubers with a Digoxigenin-LabeledcRNA probe.
Plant Disease 81:489-491.
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