Download 2006-025 (5.3) Draft Annex to ISPM 27:2006 – Aphelenchoides

International Plant Protection Convention
Draft Annex to ISPM 26 – Aphelenchoides besseyi, A. fragariae and
A. ritzemabosi (2006-025)
2006-025
Agenda item 5.3
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi (2006-025)
Status box
This is not an official part of the standard and it will be modified by the IPPC Secretariat after adoption.
Date of this document
2014-03-12
Document category
Draft new annex to ISPM 27:2006 (Diagnostic protocols for regulated pests)
Current document stage
To TPDP meeting, June 2014
Major stages
- 2006-05 SC introduced original subject: Aphelenchoides besseyi, A. fragariae
and A. ritzemabosi (2006-025)
- 2007-03 CPM-2 (2007) added subject under work programme topic:
Nematodes (2006-008)
2012-06 Lead author no longer available
2013-05 First draft in preparation, not yet seen by TPDP
2013-05 SC approved from priority 1 to priority 2
2014-03: Expert consultation
Discipline leads history
2006 Thomas SCHRODER (DE)
2009 Geraldine ANTHOINE (FR)
Consultation
on
technical The first draft of this protocol was written by:
level

Fegru Zhang, USDA, APHIS, PPQ, 389 Oyster Point Blvd., Suite 2,
South San Francisco, CA 94080

Sue Hockland, formely Nematology Unit, Food and Environment
Research Agency, Sand Hutton, York, United Kingdom

Xie Hui, Plant Nematology Laboratory, College of Natural Resources
& Environment, South China Agricultural University, Wushan Street,
Guangzhou City, Guangdong Province, 510642 China.

Rinus Knoetze, Directorate Plant Health, Department of Agriculture of
South Africa, Private Bag X5015, Stellenbosch, 7599, South Africa.
The following scientists reviewed the protocol and improved it by numerous
comments.
Main discussion points during
development of the diagnostic protocol
Notes
This is a draft document for submission for expert consultation.
2014-03 Status box last modified
International Plant Protection Convention
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
2006-025 (5.3)
CONTENTS
1. Pest Information ................................................................................................................................2
2. Taxonomic Information .....................................................................................................................5
3.
4.
Detection.........................................................................................................................................6
3.1
Symptoms ..........................................................................................................................6
3.1.1
Symptoms by Aphelenchoides besseyi ..............................................................................6
3.1.2
Symptoms by Aphelenchoides fragariae ..........................................................................6
3.2
Extraction of the nematodes ............................................................................................10
Identification.................................................................................................................................11
4.1
Morphological identification of the aphelench nemaodes ..............................................12
4.2
Identification of Aphelenchoides to species ....................................................................16
4.3
Morphological identification of A. besseyi .....................................................................22
4.4
Morphological identification of A. fragariae ..................................................................30
4.5
Morphological identification of A. ritzemabosi ..............................................................36
4.6
Molecular identification of Aphelenchoides spp. ............................................................38
5. Records ...............................................................................................................................................40
6. Contact Points for Further Information ..............................................................................................41
7. Acknowledgements ............................................................................................................................41
8. References ..........................................................................................................................................41
Endorsement
[1]
This diagnostic protocol was adopted by the Commission on Phytosanitary Measures in ----.
1. Pest Information
[2]
The majority of species within the genus Aphelenchoides Fischer, 1894 are mycetophagous, but a
small group including Aphelenchoides besseyi (Christie, 1942), A. fragariae (Ritzema Bos, 1890), and
A. ritzemabosi (Schwartz, 1911) also feed on higher plants. This group of nematodes are called
foliar/leaf and/or bud nematodes because these nematodes are common and widespread migratory
ecto- and endoparasites of leaves, buds, stems, and occasionally corms, causing crinkling, blotching
and growth retardation of the leaves, resulting in a reduction of quality and yield of many ornamental
and crop plants, such as rice, strawberry, and chrysanthemum. It is important to identify the particular
species in the infestation as the life-cycle of each species is slightly different.
[3]
Aphelenchoides besseyi is also known as the Rice Leaf Nematode because of the symptoms this
nematode causes on its major host, rice, wherever it occurs worldwide. However, it also infests
strawberries, where it is a cause of strawberry crimp disease recorded from the USA, Australia and
more latterly, Europe. Other crops recorded as infested include grasses (Panicum, Pennisetum, and
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Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
Setaria), ornamentals (such as Begonia, Chrysanthemum) and vegetables (such as Allium, Dioscorea).
(CABI, 2013).
[4]
This nematode is morphologically similar to several other Aphelenchoides and some Ditylenchus
species that infest plants; they are slender, free-living nematodes with a serpentine movement when
viewed in water under a microscope.
[5]
Rice plants susceptible to A. besseyi can be symptomless but yield or quality loss occurs when
symptoms are seen. The viability of seed infested with A. besseyi is lowered and germination is
delayed (Tamura & Kegasawa, 1959) and diseased plants have reduced vigour and height (Todd &
Atkins, 1958). The nematode is capable of withstanding desiccation, and maybe found in a quiescent
state beneath the hulls of rice grains.
[6]
In strawberry, A. besseyi may be found between leaves and buds, as with some other Aphelenchoides
spp.; all may cause distortion of the leaves which is more noticeable on newly formed leaves after
growth resumes in the spring season (Brown et al., 1993).
[7]
In rice and strawberries the nematode feeds ectoparasitically, but A. besseyi may also be endoparasitic,
as in Ficus elastica and Polianthes tuberosa, in which it causes leaf drop and leaf lesions respectively.
On Capsicum annum v. longum the infestation appears to result in rotting of the pods and premature
pod drop, similar to some fungal diseases (Hockland & Eng, 1997). In the grass Sporobolus poirettii,
the nematode stimulates growth, resulting in increased florescences.
[8]
The nematode occurs worldwide, in Africa, Asia, Australia (including Oceania), Central America and
Caribbean, Europe, North America, the Middle East, and South America. (Fortuner and Williams,
1975; CABI, 2013).
[9]
The nematode can be spread around the world when quarantine conditions are not met, on seed or
unhulled rice, but specimens have also been found in the soil of imported penjing (bonsai), probably
due then to the practice of irrigating the plants with water from paddy fields where the nematode is a
pest of rice (Hockland, personal communication). There is also a risk of spread with the practice of
using rice husks as packaging material, which has occurred on some imported consignments of plants.
[10]
Aphelenchoides fragariae, the strawberry spring dwarf nematode, is an endo- and ectoparasite of
aerial parts of plants, and is also commonly termed foliar, or bud and leaf nematode. It has an
extensive host range including over 250 plant species in 47 families and is widely distributed in
temperate and tropical regions through the world. It has been reported in Asia, Australia, Europe,
America and Oceania (EPPO, 2013).
[11]
A. fragariae is a causal agent of strawberry crimp or spring dwarf disease and can also cause serious
damage to many other agricultural and ornamental crops. A. fragariae induces economic loss of
millions dollars each year in the ornamental nursery industry (Jagdale and Grewal, 2006). This
nematode feeds on the epidermis, mesophyll and parenchyma tissues of leaves or fronds, resulting in
chlorosis or vein-delimited lesions that turn necrotic, leaf tattered and defoliate over time. A. fragariae
is a slender and vermiform nematode. The body length usually is no more than 1.0 mm with two
incisures in lateral field. Stylet is slender and short (10 to 11 μm long) with minute basal knobs.
Oesophagus has a strongly developed muscular metacarpus. Tails in both sexes are similar in form and
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2006-025 (5.3)
elongate conoid ending in a simple spike (Hunt, 1993). A. fragariae is a bisexual species which
completes its life cycle in 10-11 days at 18 o C (Siddiqi, 1975). The nematode overwinters as juveniles
and adults in soil, dry leaves and dormant buds, but not in the roots of infested plants (Jagdale and
Grewal, 2006). The overwintering nematodes migrate from soil and infested leaf debris up the stems
and petioles in water films to leaves to cause infestation when environmental conditions such as warm
temperature and high humidity favour action of the nematode. High moisture is required for the
survival and upward movement of the nematode. It migrates from infested plant tissues to uninfested
plant tissues through films of water over plant surface on a single plant, and spreads to healthy plants
via contact between healthy plant and infested leaves during the growing season; sprinkler irrigation
and rainfall also allow the nematode to move by splash dispersal from plant to plant (Jagdale and
Grewal, 2006; Kohl et al., 2010). The nematode can be transmitted over long distances in shipments of
asymptomatic infested plants.
[12]
Aphelenchoide ritzemabosi is an obligate plant parasite, inhabiting leaves, buds and growing points.
The nematode is rarely encountered in soil where it does not complete its life cycle or survive the
winter. It may feed endoparasitically on mesophyll cells of leaves or ectoparasitically on buds and
growing points (Siddiqi, 1974). The nematodes do not enter the stem tissue, but move within a water
film to reach the leaves and buds. The leaves are invaded through the stomata. The nematodes feed on
and destroy the mesophyll cells, resulting in angular leaf spot in several hosts, and also causes
dwarfing and leaf wilt.. The cells in infested areas are killed and the leaves develop brown lesions
delimited by the veins (Franklin, 1959). The nematodes exit from the leaves through the stomata and
once again migrate in the water film on the surface to infect flower buds (Southey, 1993). A.
ritzemabosi overwinters in dormant buds and growing points of chrysanthemum stools, which serve as
a source of infestation (Hesling and Wallace, 1961). A. ritzemabosi survives unfavourable conditions
through anhydrobiosis and can retain viability for some months in dried plant material. Like other
Aphelenchoides species, A. ritzemabosi can reproduce on fungi, and soil fungi may therefore
contribute to its survival in the absence of a host (Hooper and Cowland, 1986).
[13]
A. ritzemabosi was found in association with Phytophthora cryptogea on diseased gloxinia plants
(Stokes and Alfieri, 1969) and is linked together with Corynebacterium fascians in the onset of
'cauliflower' disease in strawberries (Crosse & Pitcher, 1952). Madej et al (2000) found several
plant-parasitic fungi in association with A. ritzemabosi, which increased the necrosis of
Chrysanthemum and Zinnia plants affected by the nematode.
[14]
A. ritzemabosi is a major pest of chrysanthemum in Europe, North America, New Zealand and
Australia, and has been reported on this host from several other countries (CABI/EPPO, 2000; EPPO,
2013). A. ritzemabosi, as well as A. Fragariae cause damage to strawberries in several European
countries as well as Mexico (CABI/EPPO, 2000; EPPO, 2013). The nematode is also recorded on a
wide range of ornamental and other hosts from Europe, Asia, North America, South America and
Oceania (CABI/EPPO, 2000; EPPO, 2013). The nematode was also reported as occurring in South
Africa (Wager, 1972). However, these records were made on the basis of symptoms only and the
nematodes were never positively identified taxonomically. The first report of A. ritzemabosi in South
Africa that was also morphologically identified was on Nerine bulbs in nurseries (Swart et al., 2007).
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2. Taxonomic Information
[15]
Name:
Aphelenchoides besseyi Christie, 1942
[16]
Synonyms:
Aphelenchoides oryzae Yokoo, 1948
Asteroaphelenchoides besseyi (Christie, 1942) Drozdovsky, 1967
[17]
Common names:
Preferred common name (CABI, 2013): rice leaf nematode.
International Common Names (CABI, 2013): summer crimp nematode, white
tip, white tip nematode of rice.
[18]
Name:
Aphelenchoides fragariae (Ritzema Bos, 1890) Christie, 1932
[19]
Synonyms:
Aphelenchus fragariae Ritzema Bos, 1890
Aphelenchus olesistus Ritzema Bos, 1892
Aphelenchoides olesistus (Ritzema Bos, 1892) Steiner,1932
Aphelenchus olesistus var. longicollis Schwartz,1911
Aphelenchoides olesistus var. longicollis (Schwartz,1911) Goodey, 1933
Aphelenchus pseudolesistus Goodey,1928
Aphelenchoides pseudolesistus (Goodey,1928) Goodey,1933
Aphelenchus ormerodis of Jegen, 1920 (nec of Ritzema Bos, 1891)
[20]
Common names:
Strawberry spring dwarf nematode, strawberry crimp nematode, foliar
nematode, bud and leaf nematode
[21]
Name:
Aphelenchoides ritzemabosi, (Schwartz, 1911) Steiner & Buhrer, 1932
[22]
Synonyms:
Aphelenchoides ribes (Taylor, 1917) Goodey, 1933
Aphelenchus phyllophagus Stewart, 1921
Aphelenchus ribes (Taylor, 1917) Goodey, 1923
Aphelenchus ritzema-bosi (Schwartz, 1911)
Pathoaphelenchus ritzemabosi (Schwartz, 1911) Steiner, 1932
Pseudaphelenchoides ritzemabosi (Schwartz, 1911) Drozdovski, 1967
Tylenchus ribes Taylor, 1917
[23]
Common name:
Chrysanthemum foliar nematode, leaf and bud nematode (English)
[24]
Taxonomic position: Nematoda, Aphelenchida, Aphelenchina, Aphelenchoididae.
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[25]
3.
Detection
3.1
Symptoms
2006-025 (5.3)
Aphelenchoides besseyi, A. fragariae, and A. ritzemabosi may occasionally be found in the growing
media of infested hosts, but are most commonly found in infested plant foliage, including leaf, flower,
buds, and seed heads or pods. Symptoms of infestation by these nematodes vary according to the host.
3.1.1 Symptoms by Aphelenchoides besseyi
[26]
During early growth of rice, the most conspicuous symptom caused by this nematode is the emergence
of the chlorotic tips of new leaves from the leaf sheath (Figure 1). These tips later dry and curl, whilst
the rest of the leaf may appear normal. The young leaves of infested tillers can be speckled with a
white splash pattern or have distinct chlorotic areas. Leaf margins may be distorted and wrinkled but
leaf sheaths are symptomless. A crinkling and distortion of the flag leaf enclosing the panicle; the
latter is reduced in size, as are the grains. Symptoms may be confused with calcium and magnesium
deficiency. Infested panicles are shorter, with fewer spikelets and a smaller proportion of filled grain
(Dastur, 1936; Yoshii and Yamamoto, 1951; Todd & Atkins, 1958). In severe infestations, the
shortened flag leaf is twisted and can prevent the complete extrusion of the panicle from the boot
(Yoshii & Yamamoto, 1950; Todd & Atkins, 1958). The grain is small and distorted (Todd & Atkins,
1958) and the kernel may be discoloured and cracked (Uebayashi et al., 1976). Infested plants mature
late and have sterile panicles borne on tillers produced from high nodes.
[27]
Figure 1. Symptoms caused by Aphelenchoides besseyi on rice leaves: Left and Middle: white tip.
Right: Necrotic patches and crinkled leaves. (L. from Society of Nematologists slide set, 1980; M. and
R. from John Bridge/CABI BioScience. Reproduced from the Crop Protection Compendium, 2006
Edition. © CAB International, Wallingford, UK, 2006).
[28]
On strawberries symptoms include leaf crinkling and distortion, and dwarfing of the plant with an
associated reduction in flowering. Symptoms may be confused or similar with those caused by other
Aphelenchoides species (leaf and bud nematodes) and can be found as in following figures of
symptoms caused by A. fragariae.
3.1.2 Symptoms by Aphelenchoides fragariae
[29]
A. fragariae is commonly associated with strawberry and also found in a large range of ornamental
plants, including ferns, foliage and flowering plants, and herbaceous and woody perennials (Kohl,
2011). A. fragariae is commonly found in the aerial parts of plants, corms and soil or growing media
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Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
associated with host plants. In particular, it can be detected on leaves showing discoloured mosaic or
angular spots.
[30]
Common symptoms of plants damaged by A. fragariae are chlorosis, necrosis, dwarfing of the leaves,
stems, flowers or bulbs, leaf tattering and defoliation. They are often confused with symptoms caused
by powdery mildew. Symptoms typically show vein-delimited lesions or blotches that start as lightly
chlorotic and then turn brown to black or necrotic and dry (Figure 2). Symptom expression, however,
may be highly variable due to the characteristics of host plant species and influence of environmental
conditions. The shape and pattern of the blotches is closely related to the venation pattern of leaf, such
as on Buddleja sp., Convolvulus arvensis, Phymatodes diversifolium, Salvia sp., Stachys riederi
infested leaves appearing as pale green to tan-colored or dark brown mosaic spots or angular necrotic
lesions (Figure 3) (Khan et al., 2008; Knight et al., 2002; Kohl, 2011); on Hosta leaf blotch as long
and narrow necrotic form bounded by longer veins, and in severe cases, the entire leaf turning dry and
death (Figure 4) (Zhen et al., 2012); the leaf spot symptoms on ferns showing narrow or linear patches
perpendicular to the midrib of frond corresponding with closely spaced lateral veins, as chevron-like
stripes (Figure 5) (Cobon and O’Neill, 2011); on Cyclamen sp., Begonia sp. and Andrographis
paniculata infested leaves as water soaked irregular patches later turning brown (Figure 6) (Southey,
1993; Supriadi, 2008 ). In general, the form of the blotches are more or less angular chlorotic areas in
ternate or palmate leaves with reticulate venation or with main veins radiation from the petiole-lamina
junction; infected thicker and succulent leaves initially show water-soaked irregular patches
subsequently turning necrotic without defined margins; ultimately the entire leaf dies (Richardson and
Grewal, 1993; Southey, 1993). On strawberries (Figure 7), the initial symptoms of infestation are plant
growth with stunting and deformation of buds, leaves and flowers; infested plants show malformations
including twisting and puckering of leaves, discoloured areas with hard and rough surface, undersized
leaves with crinkled edges, tight aggregation or death of crowns, reddened and stunted petioles and
flower stalks with aborted or partly aborted flowers; heavily infected plant do not produce fruit
(Siddiqi, 1975).
[31]
A. fragariae infests buds or crown, causing decay of buds, the flowers turn shrivelled, and leaves,
petioles and stems expanding from buds attacked turn misshapen, crinkled, stunted, and show brown
scars ( Richardson and Grewal, 1993; Southey, 1993). Sometimes infested plants do not exhibit
symptoms until the plant is heavily infested with nematodes, and symptom development will vary
depending on the environmental conditions and host plant species.
[32]
Figure 2: Symptoms caused by Aphelenchoides fragariae on Stachys riederi var. japonica (after
Khan et al., 2008).
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[33]
Figure 3: Symptoms of Aphelenchoides fragariae attack in Convolvulus arvensis (A), Phymatodes
diversifolium (B), Stachys riederi (C), Buddleja sp. (D), Salvia sp.(E) (after Khan et al., 2008; Knight
et al., 2002; Kohl, 2011).
[34]
Figure 4: The different degrees of symptom severity on hosta leaves caused by of Aphelenchoides
fragariae (after Zhen et al., 2012).
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[35]
Figure 5: Leaf blotch symptoms on fens (A), Pteris cretica (B) and Stenochlaena tenuiflolia infected
by Aphelenchoides fragariae (A after Cobon and O’Neill, 2011; B, C after Chizov et al., 2006).
[36]
Figure 6: Leaf blotch symptoms on Begonia sp. (A) and Andrographis paniculata (B) infected by
Aphelenchoides fragariae. (A after Department of Crop Sciences, University of Illinois at
Urbana-Champaign, 2000; B after Supriadi, 2008).
[37]
Figure 7: Strawberry plant (A, B) infected by Aphelenchoides fragariae and plant (C) not infected. A:
tight aggregation of strawberry crown with malformed leaves; B: abnormal plant growth with stunting
and deformation; C: asymptomatic plant. (after Cobon and O’Neill, 2011).
3.1.3 Symptoms by Aphelenchoides ritzemabosi
[38]
A. ritzemabosi is a major pest of chrysanthemum in Europe, North America, New Zealand and
Australia, and has been reported on this host from several other countries (Southey, 1993;
CABI/EPPO, 2000; EPPO, 2013). Infestation from the soil, dead leaves or weed hosts progresses from
the base of the plant upwards under moist conditions. Infested leaves show characteristic angular
blotches delimited by the principal veins. The discoloration progresses from translucent yellowish and
brownish green to dark brown. At a late stage, dead shriveled leaves, hanging down, extend to the top
of the plants (Figure 8). The nematodes also invade, and feed within the buds, sometimes killing the
growing point and preventing flowering, or producing malformed leaves with surface irregularities
and rough brown scars.
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[39]
Figure 8. Symptoms (interveinal necrosis) caused by Aphelenchoides ritzemabosi on Chrysanthemum
leaves. From John Bridge/CABI BioScience.
[40]
A. ritzemabosi can cause damage to strawberries as infested by A. fragariae in several European
countries as well as Mexico (Southey, 1993; CABI/EPPO, 2000; EPPO, 2013). Damage is most
noticeable on newly formed leaves, which become puckered and distorted and may show rough,
greyish feeding areas near the base of the main veins. The 'cauliflower' disease of strawberry, resulting
in the continued production of axillary buds on affected crown was experimentally induced in
strawberry runners through co-inoculation of A. ritzemabosi and Corynebacterium fascians by Crosse
and Pitcher (1952).
[41]
A. ritzemabosi causes polygonal blotches on leaves of infected plants that are bound by veins, similar
to symptoms on chrysanthemum (Johnson, 1998; Shepherd & Barker, 1990). Species of
Aphelenchoides have been reported to damage tobacco in France, Germany, China, Pakistan, Brazil
and Chile.
[42]
This nematode also infests many herbaceous plants; most show the typical interveinal leaf blotches
and distortions of the upper leaves due to bud infestation. A. ritzemabosi is also associated with death
of lower leaves and malformed growth of shoots in lavender, caused by bud infestation. Woody plants.
Like Buddleia are also attacked, causing death of buds and leaf distortions. Attacked violets are
stunted, and affected leaves curl down, wither and die; under surfaces of leaves showed typical
water-soaked blotches (Thomas, 1968; Southey, 1993). Stunting and shoot blindness occurred on
attacked Crassula coccinia (Atkinson, 1964). The presence of A. ritzemabosi in stem tissues in
combined infestations with Ditylenchus dipsaci, show discoloration caused by feeding of A.
ritzemabosi after cell separation by the action of D. dipsaci.
3.2
Extraction of the nematodes
3.2.1 Direct examination
[43]
In leaves infested with Aphelenchoides besseyi, A. fragariae, or A. ritzemabosi, nematodes can be
detected by inspecting the small young cut leaves covered tap water in a Petri dish under a
stereomicroscope (the nematodes will swim into the water within 30 minutes if there is a heavy
infestation).
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3.2.2 Extraction from plant materials
[44]
Aphelenchoides besseyi, A. fragariae, or A. ritzemabosi can be extracted from plant tissues, soil or
growing medium with suspected infestation using the Baermann funnel technique, Modified
Baerman-tray method (Hooper and Evans, 1993, an adapted sugar-flotation method (Coolen &
D’Herde, 1972): or the mistifier technique (Hooper et al., 2005). The presence of the nematode on
plant material can also be extracted using the water soaking method that was more efficient than the
Baermann funnel technique (Zhen et al., 2012). These extraction methods should be conducted for 48
hours at room temperature to detect low levels of infestation. Any plant material to be tested should be
cut into pieces or sliced before extracting for increasing efficacy of extraction.
[45]
More refined detection methods are required for A. besseyi in rice seeds. The presence of the nematode
on rice seed has been determined by following the EPPO Quarantine Procedure no. 38 (EPPO, 1992).
This method uses whole seeds, but recent work by Moretti et al., (1999), reported by the EPPO
Reporting Service (99/086), recommends the use of rice chaff or hull as an alternative testing material.
Hoshino & Togashi (2002) have also described a mass extraction method said to be more efficient
than the Baermann funnel technique. More recently, laboratories wishing to adopt universally tested
methods have adopted the Seed Health Testing Method 7-025: Detection of Aphelenchoides besseyi on
Oryza sativa, produced by the International Seed Testing Association (Remeeus & Pelazza, 2013).
[46]
Under a stereoscopic microscope, the stylet-bearing nematodes with a well-demarcated large median
pharyngeal bulb can be transferred with a pipette or a needle from all the nematodes isolated in a small
Petri dish to a glass slide for microscopic examination.
4.
Identification
[47]
Although Aphelenchoides can be identified to species level based on the morphological examination,
this method is only possible for the adult specimens, as is the case for most other plant-parasitic
nematode species. The presence of immature stages in samples can also provide some important
additional information such as confirming their development on host plants, and to provide genetic
material for DNA analysis. The morphological characters of Aphelenchoides need to be carefully
examined by using a high power microscope, with at least x1000 magnification for use with
immersion oil, or by using the scanning electron microscope (SEM) for precise species level
identification. Since the nematodes of Aphelenchoides are very difficult to identify to species level just
using morphological characters, molecular diagnostic tools have also been developed for the
identification of Aphelenchoides and these methods may be used to minimise the possible error and to
support the morphological identification of these species (Ibrahim 1994a, 1994b).
[48]
Molecular methods can be applied to identification of all life stages including the immature stages and
may be used when there is a low level of infestation or when adult specimens are atypical or damaged.
The specificity of currently available molecular assays may be limited as they have generally been
developed and evaluated using a restricted number of species and populations from different
geographic regions. The use of DNA sequencing shows promise for providing a specified degree of
confidence in identification, but only peer-reviewed databases, such as QBank, should be consulted
(Bonants et al., 2013).
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4.1
2006-025 (5.3)
Morphological identification of the aphelench nemaodes
4.1.1 Preparation of aphelenchs for morphological identification
[49]
Individual nematodes of Aphelenchoides species can be picked from the extract by using any of the
above-mentioned extraction methods and are collected into a drop of water on a slide. These
nematodes are slowly heated (to approximately 60 o C) until the nematodes become immobile (Hooper
et al., 2005). The body of these nematodes killed by gentle heat is almost straight. The nematodes can
be sealed on the slide with wax during this process, or they can be placed in a drop of fixative before
sealing with wax. There are some differences in the appearance of water and fixed specimens, with the
former being preferable, but in fixed preparations some features such as stylets become more distinct.
Ideally about 5 females and 5 males should be placed on a slide for examination. At least one female
and one male should be prepared for this examination if there are no enough numbers of available
specimens.
[50]
Under a stereomicroscope the round, relatively large median bulb is a distinctive feature of nematodes
belonging to the Order Aphelenchida. These nematodes are vermiform, with most species appearing to
be of stout structure and slow-moving. However, the few economically important species such as A.
besseyi, A. fragariae, or A ritsemabosi tend to be of slim build, pale appearance, relatively long when
compared to most other Aphelenchoides species and good swimmers (with a serpentine motion) in
water. Of course there are always exceptions to the rule, e.g. A. blastophthorus, a pest of ornamental
plants and strawberries that rarely swims well and is of stouter structure.
[51]
For SEM studies, killed and fixed nematodes are washed with distilled water or ultrasonic vibrator to
remove adhering detritus, and then treated with glutaraldehyde and dosmium tetraoxide or other
solution, dehydrated in graded ethanol series, critical point dried, sputter coated with gold, and
observed with scanning electron microscope (Shepherd and Clark, 1970; Eisenback, 1991; Mitkowski
et al., 2002).
4.1.2 Identification of the order Aphelenchida
[52]
Nematodes of the order Aphelenchida show the following characteristics (Hunt, 1993): Body small to
long (0.2-2.5mm), vermiform, rarely obese except in some insect parasites. Cuticle thin, usually finely
annulated and with lateral fields marked by 0 to more than 12 incisures. Cephalic region low, rounded,
continuous or offset and with weak or moderate sclerotization. Four submedian cephalic papillae and
six readily visible labial papillae (plus possibly six, more obscure, labial papillae) around the oral
aperture. Amphidial apertures oval, pore-like, dorsosublateral on labial region. Stylet always
present, length usually 10-20μm, but exceptionally up to 185μm; conus usually shorter than the shaft,
but much longer in certain insect ectoparasitic forms. Basal swellings or knobs usually weakly
developed or entirely absent. Oesophagus comprising a narrow, cylindrical procorpus, a
strongly-developed, offset, ovoid to rounded rectangular median bulb with crescentic valve
plates and well-developed oesophageal glands forming a dorsally overlapping lobe in all genera
apart from Paraphelenchus where the glands are small and enclosed in a non-overlapping basal bulb.
All three gland orifi (i.e. including the dorsal gland orifice) located within the median bulb.
Isthmus usually short or absent. Nerve ring circumoesophageal or circumintestinal. Intestine cellular
with distinct lumen. Rectum usually distinct except in some insect associates. Anus a broad
transverse slit with an overhanging anterior lip, but absent or degenerate in some insect parasites or
Page 12 of 46
International Plant Protection Convention
2006-025 (5.3)
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
associates. Vulva posterior at 60-98%, usually in the form of a transverse slit or, exceptionally, an
oval pore (Aphelenchus). Genital tract monoprodelphic, usually outstretched, but occasionally
with a double flexure. Spermatheca axial, if present. Post vulval sac usually present and may act as a
spermatheca. Male genital system monorchic, outstretched. Sperm large, rounded, arranged in one or
two rows in the gonoduct. Spicules typically rosethorn-shaped with prominent apex and rostrum,
or derived therefrom, but elongate and cephalated in Aphelenchus and Paraphelenchus.
Gubernaculum usually absent, but well-developed and elongate in Aphelenchus and Paraphelenchus.
Bursa usually absent, but a true peloderan bursa with supporting ribs is present in Aphelenchus only,
although some genera may have a terminal flap of cuticle (= bursa). Usually three pairs of caudal
papillae present, but may number as few as two or as many as five pairs.
[53]
The nematodes of Aphelenchida can be reliably identified with key characteristics, which include the
protrusible stylet; oesophagus consisting of a thinner procorpus, a very prominent, ovoid to rounded
rectangular median bulb with crescentic valve plates and well developed oesophageal glands; the
median bulb very large, often appears nearly as wide as the diameter of the body, and containing
orifice of the dorsal oesophageal gland.
4.1.3 Identification of the family Aphelenchoididae
[54]
The family Aphelenchoididae is characterized by large metacorpus, oesophageal glands usually not
enclosed in a bulb (overlapping). Dorsal oesophageal gland opens into metacorpus. Males have
caudal papillae.
[55]
Aphelenchoides besseyi, A. fragariae, and A. ritzemabosi belong to the family Aphelenchoididae
which share the morphological characteristics outlined in Table 1.
[56]
Table 1: Family Aphelenchoididae shared main morphological characteristics
Body part
Characteristic
Body form
vermiform, no swollen
Lateral field
with four or fewer incisures
Stylet
slender, with narrow lumen and usually with small basal knobs or swellings
Oesophagus
Isthmus rudimentary or absent, nerve ring circumoesophageal, oesophageal glands
lobe-like and long dorsally overlapping intestine
Post-uterine sac
usually present
Spicule
rose-thorn shaped or derived therefrom
Adanal bursa
Absent
Gubernaculum
Absent
Tail shape
both sexes similar, conoid, with pointed or rounded, often mucronate terminus
4.1.4 Identification of the genus Aphelenchoides
[57]
Hunt (1993) and Andrássy (2007) respectively produced a dichotomous key for the identification of
genera within the family Aphelenchoididae. Aphelenchoides is the genus with the most species in the
International Plant Protection Convention
Page 13 of 46
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
2006-025 (5.3)
Aphelenchida. Over 250 species have been described under this genus or placed in it from other
genera by now. Some members of these species, however, are poorly characterized or have been
transferred to subsequently established genera, so that the nominal species belonging to
Aphelenchoides are no more than 200 at present (Andrássy, 2007).
[58]
As with many other nematode genera, Aphelenchoides species are morphologically very similar and
the genus of Aphelenchoides can be distinguished by following morphological characteristics.
[59]
Diagnostic characteristics of Aphelenchoides spp. (Figure 9):
[60]
Body Length: generally from 0.2 to 1.3mm, but most commonly 0.4 to 0.8 mm.
[61]
Heat relaxed females: die straight to ventrally arcuate (Figure 9A).
[62]
Heat relaxed males: assume a ‘walking-stick’ shape with the tail region curled ventrally (Figure 9B).
[63]
Cuticle: finely annulated, lateral field with two to four (rarely six) incisures (Figure 9D).
[64]
Stylet: Very difficult to see under low power. Under high power it varies from clearly discernible to
very faint. Generally about 10-12 microns long. Similarly basal knobs or swellings are sometimes
clear but often indistinct. Unfortunately, measurements of these knobs have rarely been made.
[65]
Oesophagus: Oesophageal procorpus long and slender; metacorpus (medial bulb) well developed,
spherical to rounded–rectangular, with central valve plates; oesophageal gland lobe long dorsally
overlapping intestine (Figure 9C).
[66]
Vulva: typically post-median, usually between 60-75% of the body length.
[67]
Ovary: monoprodelphic, typically outstretched, but may be flexed.
[68]
Post-vulval sac: almost always present.
[69]
Tail shape: conoid to variable (Figure 9EF), in male more strongly curved ventrally and papillate
variable.
[70]
Tail terminus: without or with one to more mucros (Figure 9GH), a mucro is defined as a structure at
the end of the tail terminus. Can only truly be discerned at x1000 with oil immersion. The author
considers that the presence or absence of mucros, and the shapes they portray, can be used to
distinguish species, and is a key element in the identification of Aphelenchoides besseyi, A.fragariae,
and A. ritzemabosi.
[71]
Spicules: well developed, thorn shaped, thorn-shaped, paired and separate.
[72]
Bursa: absent.
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Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
[73]
Figure 9: Aphelenchoides spp.: A. Female; B. Male; C. Female anterior end; D. Lateral field; E.
Female tail; F. Male tail; G. Female tail terminal mucro; H. Male tail terminal mucro. (A, B, E after
Wang et al., 2013; D, G after Deimi et al., 2006; H after Yu and Tsay, 2003; C, F : courtesy Z. F.
Yang and H. Xie, South China Agricultural University).
[74]
Aphelenchoides spp. can be distinguished from other nematode species encountered in soil and plant
tissue by using the key in Table 2.
International Plant Protection Convention
Page 15 of 46
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
[75]
2006-025 (5.3)
Table 2: Key to distinguish Aphelenchoides spp. from other nematode species in soil and plant tissue
1
2
Stylet present
2
Stylet absent
NAS*
Three-part oesophagus with corpus (a cylindrical procorpus followed by a valvulated
3
metacorpus), slender isthmus and glandular basal bulb
Two-part oesophagus, anterior part slender, posterior part expanded glandular and
NAS
muscular
3
Dorsal oesophageal gland outlet in metacorpus; metacarpus very large, often appears
4
nearly as wide as the diameter of the body
Dorsal oesophageal gland outlet in procorpus behind stylet knobs; metacarpus moderate
NAS
to reduced in size (less than three fourths body width)
4
Oesophageal glands lobe-like, long dorsally overlapping intestine
5
Oesophageal glands pyriform, no overlapping intestine; or oesophageal glands lobe-like,
NAS
ventrally overlapping intestine
5
Lateral fields with four or fewer incisures; stylet with basal knobs or swellings; female tail
6
conoid or elongate conoid or convex-conoid or subcylindrical to a pointed or narrowly
rounded terminus; male spicules thick, thorn shaped; adanal bursa absent
NAS
Lateral fields with six or more incisures; stylet without basal knobs; female tail short ,
sub-cylindroid and with broadly rounded terminus; male spicules slender, tylenchoid;
adanal bursa present
6
Tails of both sexes short, mostly less than four times anal body width
7
Tails of both sexes elongate to filiform, predominantly more than four times anal body
NAS
width
7
Stylet slender, often about 10-12µm and usually less than 20µm; vulval flap absent, male
Aphelenchoides
without small bursa-like flap at tail tip
Not with the above combination of characters
NAS
*NAS, not Aphelenchoides species
[76]
The majority of Aphelenchoides species are free-living, saprophagous forms occurring in soil,
decaying plant material, galleries of wood-boring beetles, etc. and probably feeding on fungal hyphae.
A few species of the genus parasitize plants and yet may live on fungi. The plant infesting
Aphelenchoides species, called bud and leaf nematodes, include A. besseyi, A. blastophthorus, A.
fragariae, A. ritzemabosi and A. subtenuis, which live in above-ground parts of plants. They can be
distinguished from other species of the genus Aphelenchoides by the slender body and the more
posterior position of the hemizonid: six to ten annules behind the excretory pore (vs. one to three
annules) (Thorne, 1961).
4.2
Identification of Aphelenchoides to species
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International Plant Protection Convention
2006-025 (5.3)
[77]
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
The identification of species in this genus is complex, and requires a systematic approach. It is
generally agreed that a combination of morphological and biochemical methods is required for the
most reliable identification. The first step in diagnosis is to record and measure the critical
morphological features on as many female specimens as are available, ideally 20. In practice, far fewer
adult specimens are usually available, and in such cases the nematologist should prepare the
specimen(s) with great care to avoid spoiling the few features available. Males are not included in the
keys presented here, but the shape and size of the spicules may assist in confirming the final
identification.
4.2.1 Morphological characters of selected Aphelenchoides species
[78]
A table compiles morphological characters for selected Aphelenchoides species (EPPO, 2004) and is
reproduced in Table 3.
[79]
Table 3: Morphological characters of selected Aphelenchoides species (taken from EPPO, 2004)
Species
mucro
length of
shape
pvs
A. aligarhiensis star
tail shape
position of excretory a
b
pore and nerve ring
oid
anterior to nerve ring
elongate
not possible to advise
stylet
L.L. c
(mm)
×5.5 body elongate-con excretory pore slightly 25–35 7.0–9.0
width,
Length
0.50–0.70 10
4
13-22
extending
to more
than
50% of v-a
distance
A. andrassyi
star
×3 vulval
23–28 3.2–3.9
0.39–0.44 9.0–10.0 3
.0
body width conoid
A.
star
conoid
body width
asterocaudatus
A.
×2 vulval
star
×1 vulval
excretory pore posterior
to nerve ring
conoid
body width
of lower edge of nerve
us
or less
ring
star
2.5–3.5
conoid
9.6
0.62
12
2
16
4
10.9–1
24.6
excretory pore at level 32–39 5.5–9.5
asteromucronat
A. besseyi
6.0–12
0.39–0.54 9
4.5
excretory pore usually 32–42 10.2–11. 0.66–0.75 10–12.5 4
anal body
near the anterior edge
width but
of the nerve ring
17–21
4
less than
33% v-a
distance
A.
single
approx.
conoid
blastophthorus terminal 50% v-a
mucro
excretory pore approx. 32–47 9.3–11
0.68–0.9
15–19.5 4
16–21
opposite nerve ring
distance
International Plant Protection Convention
Page 17 of 46
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
A. brevistylus
star
33–66%
conoid
v-a
2006-025 (5.3)
excretory pore anterior 29.1–3 8.1–10.5 0.39–0.63 6.0–8.0
2
11.1–1
5.7
to nerve ring
5.0
excretory pore level
45–63 8–15
0.45–0.80 10–11
2
12–20
29–39 n/a
0.46–0.61 11.5–12. 4
14–18
distance
A. fragariae
no mucro more than elongate
50% v-a
conoid
distance
A. goodeyi
star
×3 vulval
with or close behind
nerve ring
convex-conoi excretory pore close
body width d
behind nerve ring, level
5
with its hind edge
A. hylurgi
A. jonesi
star
star
excretory pore slightly 26.6
width
posterior to nerve ring
ure
excretory pore opposite 20–28 9.3–11.0 0.72–0.99 11–14
4
16–22
9.5-10
4
15-17
0.597
12.8
3
17
0.77-1.2
12
4
18-24
nearly ×2
conoid
body widths
10.2
0.57
13
obsc 14.7
×1.5 body conoid
nerve ring
long.
Nearly 50%
v-a
distance
star
A. lichenicola
50% or
elongate-con excretory pore opposite 32-43 7.0-10.5 0.53-0.69
more v-a
oid
distance
or close to posterior
margins of the nerve
ring
A. nonveilleri
star
×3 body
conoid
not known
31
n/a
elongate
excretory pore 0.5–2
40-45 10-13
conoid
body widths posterior
width
A ritzemabosi
tail
50% or
terminus more the
invariably v-a
to nerve ring
ending in distance
a box-like
structure
which
has two
to four
minute
processe
s
pointing
posteriorl
y
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International Plant Protection Convention
2006-025 (5.3)
A. siddiqii
star
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
×0.5–1.0
subcylindrical excretory pore usually 26.66– 7.14–9.7 0.37–0.70 11–12.5 4
body width
opposite the posterior
38.88
14.11–
19.64
2
margins of the nerve
ring, sometimes
reaching near its
anterior margins
A. silvester
star
no
convex-conoi no information
0.48-0.56
9.5-10
4
15-16
30-36.9 7.9-10.2 0.48-0.76
10-11
2
13.0-1
37-38 8.0-9.7
information d
A. unisexus
star
20%–33% conoid
excretory pore at the
v-a
level of the posterior
distance
margin of the nerve
7.5
ring, or just posterior to
it
A. wallacei
star
×2 body
convex-conoi excretory pore anterior 22-23 7.8-8.5
widths long d
International Plant Protection Convention
0.69-0.73
13.5-14
4
15-17
to the nerve ring
Page 19 of 46
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
2006-025 (5.3)
4.2.2 Keys to provisional identification of species
[80]
Sanwal (1961) produced a dichotomous key to 35 Aphelenchoides species that were recognised at the
time, but to date over 140 species have been described. Fortuner (1970) devised a dichotomous key to
Aphelenchoides species with star-shaped mucros, but the author considers this is no longer valid,
because
[81]
(i) A. appendurus has a leaf-like appendage, not a star-shaped mucro, and the original author did not
consider it similar to any Aphelenchoides which had the latter character,
[82]
(ii) A. ritzemabosi has a box-like appendage with minute processes directed posteriorly, not a
star-shaped mucro, and
[83]
(iii)
A. silvester, described in 1968, was not included, and neither are six other species with
star-shaped mucros described since the key by Fortuner was published.
[84]
For this protocol, which concentrates on 3 species, a dichotomous and a polytomous key are offered
that should allow the reader to proceed reliably to the relevant specialist section. This is because
frequently it is not possible to determine a singular character in a short key that is required for
identification, whilst a polykey allows the combination of a range of characters for a provisional
identification.
[85]
As A. besseyi, A. fragariae and A. ritzemabosi can all occur in a wide range of habitats, including
occasionally planting media, one has to consider all Aphelenchoides nematodes that may be also be
found. Unfortunately, many of these nematodes are difficult to identify because there is little to
distinguish between them, a problem that is not facilitated by poor descriptions of the species
themselves. Indeed, several authors have improved the original descriptions for these species. In
addition, studies on Aphelelchoides species have shown the degree of variation in measurements made
on populations from different hosts (Table 4).
[86]
As with all identifications involving the use of morphological characters, the combination of several
key features is the key to a positive diagnosis. In the polykey there is some overlap of codes, and users
are advised to refer to original descriptions if in doubt about a diagnosis, or refer to the database
(Table 4) for further guidance.
4.2.2.1
[87]
Dichotomous key for Aphelenchoides species
Table 4: Key to distinguish Aphelenchoides besseyi, A. fragariae, and A. ritzemabosi from other 3 species
1
2
3
4
Post-vulval sac length more than one-third distance between the vulva and the anus
2
Post-vulval sac length less than one-third distance between the vulva and the anus
A. besseyi
lateral field with three to four incisures
3
lateral field with two incisures
A. fragariae
tail terminus with a single mucro
4
tail terminus with two to four mucro
A. ritzemabosi
stylet length less than 13 µm
5
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International Plant Protection Convention
2006-025 (5.3)
5
4.2.2.2
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
stylet length 14-19 µm
A. blastophthorus
body 0.87-1.15 mm long, more slender (a=44-57)
A. subtenuis
body 0.45-0.62 mm long, less slender (a=26-33)
A. saprophilus
Polytomous key for Aphelenchoides species
[88]
Polytomous keys allow the easy addition of new species into an identification procedure. Several
important features of species related to A. besseyi have been selected to produce a small polytomous
key (Table 5). The selected features have been given codes. Unfortunately it has only been feasible to
convert six characters into codes, namely the shape of the mucro (A), pvs length (B), tail shape (C),
stylet length (D), the number of lateral lines (E) and the relative positions of the excretory pore and
nerve ring (F). Supplementary drawings of the types of mucros and tail shapes are provided in Fig. 10
to aid categorisation.
[89]
Each suspect specimen should therefore be examined and assigned a set of codes according to the
following categorisation:
[90]
Table 5: From A. BESSEYI Protocol (after Hockland (EPPO, 2004)).
Species/Code
A
B
C
D
E
F
A. besseyi
1
1
1
1
1
1
A. hylurgi
1
1
1
1
4
3
A. unisexus
1
1
1/3
1
3
3
A. asteromucronatus
1
1
1/3
2
1
3
A. siddiqii
1
1
3/4
1
1
1/2/3
A. asterocaudatus
1
1/2
1
1
3
3
A. andrassyi
1
1/2
2/3
2
4
A. wallacei
1
1/2
3
3
1
1
A. goodeyi
1
2
1
1
1
3
A. lichenicola
1
2
1
1/2
1
2/3
A. silvester
1
2
1
1/2
1
4
International Plant Protection Convention
1/2
Page 21 of 46
Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
A. fujianensis*
1
2
1
A. jonesi
1
2
1
A. brevistylus
1
2
1/2
A. aligarhiensis
1
2
A. blastophthorus
2
A. ritzemabosi
A. fragariae
1/3
1
1/2
1
2
2
3
1
2/3
1
1
1
2
1/2
3
1
2
4
2
2
1
1
3
2/4
2
2
1
3
2/3
1/3
2006-025 (5.3)
* The codes for A. fujianensis have been assigned by the authors Zuo et al. (2010).
[91]
n.b. A. nonveilleri and A. saprophilus, mentioned in previous editions of this key, are now considered
by the author to be species indeterminatae.
4.3
Morphological identification of A. besseyi
4.3.1 Identification using morphological keys
[92]
The author has therefore attempted to reduce the number of comparisons that need to be made by
selecting only those Aphelenchoides species that also have a star-shaped mucro, together with those
pest species that might also be encountered in foliage. a dichotomous key are included for ring testing.
It should be noted that the following procedure relies heavily on the original descriptions and drawings
of species which at times can appear contradictory. For example, the tail shape for A. aligarhiensis is
described as elongate-conoid, but the accompanying drawing does not infer this. There is also no
accompanying value for ‘c’’, which is an indicator of tail shape, being the value of tail length divided
by body width at anus. Similarly, the excretory pore for A. jonesi is said to be opposite the nerve ring,
but the accompanying drawing infers that it is posterior to the nerve ring. In such cases the written
description has been the one included in this protocol. Where possible, original data has been
supplemented by additional published information for the most commonly encountered species.
4.3.2 The dichotomous key for A. besseyi
[93]
[94]
[95]
The author offers a short dichotomous key especially for this protocol. Only characters from female
nematodes have been considered. This is complemented by Fig. 10, showing critical features, and
Table 2, giving more details of those Aphelenchoides species that also have a star-shaped mucro,
together with those pest species that might also be encountered in foliage.
1. Nematode has a star-shaped mucro.................................................…………………….….............2
Nematode does not have a star-shaped mucro.....................................…………………….not A. besseyi
2. Nematode has a pvs up to 33% of the length of the distance from the vulva to the
anus.................................................................................………..............................................................3
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International Plant Protection Convention
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Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
Nematode has a pvs more than 33% of the length of the distance from the vulva to the
anus………………………........................A. aligarhiensis, A. brevistylus, A. fujianensis, A. lichenicola
[96]
[97]
[98]
3. Tail shape is conoid, or elongate conoid.............................................................……………………..4
Tail shape is sub-cylindrical...........................................................................................………A. siddiqii
4. Stylet length is in the range 10μm – 12.5μm…………………………………………………………5
Stylet length is outside this range…………………………A. asteromucronatus, A. hylurgi, A. wallacei
5. Nematode has four lateral lines………………………………………………………………………6
Less or more than four lateral lines……………………….A. andrassyi, A. asterocaudatus, A. unisexus
[99]
6. Excretory pore anterior to, or level with, the anterior level of the nerve ring…………...….A. besseyi
Excretory pore level with the nerve ring………………………………A. goodeyi, A. jonesi, A. silvester
[100]
A check on the probable identity of the nematode should be made with reference to Table 2 and the
relevant species description.
[101]
mucro shape
1 = star (Fig 10 A a-f)
2 = single terminal mucro (Fig 10 A g-m)
3 = bifurcate (Fig 10 A n)
4 = other (Fig 10 A o-t)
5 = no mucro (Fig 10 A u-v)
See Figure 10. Following text is modified from Hunt, 1993.
[102]
Fig 10 A. Tail terminus types of Aphelenchoides species.
(a) - (f): star shape (a) A. aligarhiensis b. A. asterocaudatus c. A. besseyi d. A. goodeyi (all scale
bars 10μm) e and f. A. nonveilleri (x 1100 and x2200 respectively)
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[103]
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Fig. 10 A. Tail terminus types of Aphelenchoides species (continued).
(g) - (m): single terminal mucro; g. A. richardsoni h. A. nechaleos i. A. vaughani j. A. tsalolikhini
l. and m. A. submersus (n): bifurcate: A. bicaudatus (all scale bars 10μm)
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Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
Fig. 10 A. Drawings of the tail terminus types of Aphelenchoides species (continued).
(o) - (t): other. o.p.q. A. ritzemabosi r. A. sphaerocephalus s. A. gynotylurus t. A.
helicosoma (scale bars all 10μm) (u) – (v): no mucro u. A. microstylus (scale bar 10μm) v.
A. obtusus (x1,250)
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[105]
B. length of the post-vulval sac (pvs)
1 = pvs length 33% or less the distance between the vulva and the anus
2 = pvs length more than 33% the distance between the vulva and the anus
3 = no pvs
[106]
C. tail shape
2006-025 (5.3)
1 = conoid: shape of a cone, with both sides of the tail surface tapering at an equal angle to the tail tip.
Total length not exceeding x 5 anal body width (Fig. 10 B 1).
2 = elongate-conoid: an elongated cone, with a length x5 or more anal body widths long (Fig. 10 B
2).
3 = dorsally convex-conoid: tail shape that at its first appearance is curved ventrally. The dorsal side
of the tail is curved in a convex manner before it joins with the ventral surface. The ventral surface is
usually concave, but from some viewpoints may appear straight. It may be any length (Fig. 10 B 3).
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4 = sub-cylindroid: both sides of the tail appear to run parallel for most of their length, and end with
a hemispherical or sub-hemispherical tail tip (Fig. 10 B 4).
[107]
Fig. 10 B. Drawings of tail shapes in Aphelenchoides species.
1. conoid: a. A. blastophthorus
2. elongate-conoid: b. A. andrassyi (no scale bar) c. A. chalonus
3. dorsally convex-conoid: d. A. fluviatilis (x 1100) e. A. franklini
4. sub-cylindroid: f. A. subtenuis
(scale bars = 10μm)
[108]
D. stylet length (μm)
1 = 10-13
2 = less than 10
3 = more than 13
[109]
E. L.L.s (number of)
1 = 4 lines
2 = 3 lines
3 = 2 lines
4 = unknown
[110]
F. Relative position of the excretory pore and nerve ring
1 = excretory pore anterior to, or level with, the anterior level of the nerve ring (Fig. 10 C 1).
2 = excretory pore level with the nerve ring (Fig. 10 C 2).
3 = excretory pore posterior to, or opposite the posterior level of the nerve ring (Fig 10 C 3).
[111]
Fig. 10 C. Positions of the excretory pore relative to the nerve ring in Aphelenchoides species.
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[112]
[113]
Excretory pore is anterior to, or level with the anterior edge of the nerve ring
a.
A. longiurus
b.
A. blastophthorus
2. Excretory pore is level with the nerve ring (from behind the anterior point to in front of the posterior
point)
c.
[114]
[115]
2006-025 (5.3)
A. cibolensis
3. Excretory pore is level with the posterior edge of the nerve ring (d), or posterior to it (e).
d.
A. arcticus
e.
A. ritzemabosi (scale bar = 10μm)
The set of codes obtained should then be compared with those set out in Table 4, which should allow a
provisional diagnosis to be made.
4.3.3 Morphological characteristics of Aphelenchoides besseyi
[116]
Figure 11 Morphology of Aphelenchoides besseyi.
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Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
Female: Body slender, straight to slightly arcuate ventrally when relaxed. Cephalic region rounded,
unstriated, slightly offset and wider than body at lip base. Lateral fields about one-fourth as wide as
body, with four incisures. Median oesophageal bulb oval, with a distinct valvular apparatus slightly
behind its centre. Excretory pore usually near anterior edge of nerve ring. Post-vulval sac narrow,
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2006-025 (5.3)
inconspicuous, not containing sperms, 2.5-3.5 times anal body width but less than one-third the
distance from the vulva to the anus. Tail conoid 3.5-5 anal body widths long. Terminus bearing a
mucro of diverse shape with three to four pointed processes.
[118]
Male: About as numerous as females. Posterior end of body curved by about 180º in relaxed
specimens. Tail conoid, with terminal mucro with two to four pointed processes. Spicules typical of
the genus except that the proximal end lacks an apex and has only a moderately developed rostrum.
4.3.4 Comparison with similar species
[119]
The star-shaped mucro is a very distinctive feature possessed by at least 16 other species described in
the genus (this change is to accommodate the likely addition of new species in the near future. These
species can be distinguished from A. besseyi by following the guidance given in the keys. Those
species that remain indistinguishable apart from the position of the excretory pore, a feature often
difficult to see, can be further distinguished by additional features: A. goodeyi has a convex-conoid tail
and is generally shorter in its body length (0.46-0.61mm compared to 0.57-0.88mm for A. besseyi), A.
jonesi generally has a lower value for ‘a’ (20-28, compared with 26-58) and a slightly higher range of
values for its stylet length (11μm-14μm, compared to 10μm-12.5μm), A. silvester has a
convex-conoid tail shape, a slightly lower value for ‘b’ (8-9.7, compared with 9-13.1) and is generally
shorter in body length (0.48mm-0.56mm, compared with 0.57mm-0.88mm). Males are commonly
found when A. besseyi is numerous, but they have not been described for A. goodeyi, A. jonesi or A.
silvester.
[120]
Apart from the shape of the mucro, A. besseyi differs from other plant-parasitic species of this genus
occurring in strawberries (A. blastophthorus, A. fragariae and A. ritzemabosi) as follows: A. besseyi
has a post-vulval sac that is always less than a third of the distance from the vulva to the anus, whereas
those of the other species are longer than this; the tail of A. besseyi has a conoid shape, similar to A.
blastophthorus, but shorter than that of A. fragariae and A. ritzemabosi, which tend to be
elongate-conoid; the excretory pore is usually positioned near the anterior edge of the nerve ring in A.
besseyi, whereas in the other species it is either level with or posterior to the nerve ring; the spicules of
A. besseyi are distinctive in that the proximal end lacks a dorsal process (or apex) and only a
moderately developed ventral one (rostrum), whilst those of A. blastophthorus are comparatively large
for the genus, have a rather stout dorsal limb which is characteristically flattened about mid-way
along its arch, its distal end is curved ventrally to give it a hooked or knobbed appearance, and the
apex and rostrum are pronounced structures, those of A. fragariae have a moderately developed apex
and rostrum, whilst the smoothly curved spicules of A. ritzemabosi seem to lack a dorsal or ventral
process at all.
4.4
Morphological identification of A. fragariae
4.4.1 Morphological characteristics of A. fragariae (Figure 12)
[121]
Allen (1952), Siddiqi (1975) and Hunt (1993) all provide detailed descriptions of A. fragariae (Figure
11). This description was modified from Hunt (1993).
[122]
Female. Body slender (a=45-70), straight to arcuate ventrally when relaxed. Cuticle finely annulated,
lateral field with two incisures. Cephalic region almost continuous with body, appears smooth in
microscope, and 4-5 annuli visible in SEM (Khan et al., 2007; 2008). Stylet slender, about 8-14 µm
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long, often 10-11µm; conus and shaft nearly equal in length; basal knobs minute but distinct.
Oesophagus typical of the genus, median bulb oval and highly muscular with central valve plates,
oesophageal gland lobs dorsally overlapping intestine two to four body widths long. Nerve ring
encircling isthmus near its base, about one body width behind median bulb. Excretory pore level with
or close behind nerve ring. Genital tract monoprodelphic, outstretched, with oocytes in a single file,
never reaching to oesophagus. Post-vulval sac long, extending more than half vulva-anus distance.
Tail elongate conoid with a single simple spike or minute mucro at tail tip.
[123]
Male. Abundant. Essentially similar to female in general morphology. Tail arcuate through 45° to 90°
when relaxed, not sharply curved like a hook, with a simple terminal spine. Three pairs of caudal
papillae present. Spicules rose thorn-shaped with moderately developed apex and rostrum, dorsal limb
10-19 µm long.
[124]
The key diagnostic features to distinguish A. fragariae from the other known species of the
Aphelenchoides are:
-
body 0.40-1.0 mm long , very slender (a=45-70) ( Figure 12 EF )
-
stylet slender, about 8-14 µm long, with distinct basal knobs ( Figure 12 AB)
-
tail elongate conoid with a single simple spike or mucro at tail tip ( Figure 12 GIMPQ)
-
post-vulval sac extending more than half vulva-anus distance ( Figure 12 F)
-
excretory pore level with or close behind nerve ring
-
lateral field with two incisures ( Figure 12 HO)
-
males common, spicules dorsal limb 14-17 µm long ( Figure 12 L).
4.4.2 Comparison with similar species
[125]
Aphelenchoides is a large genus, many species of which have very similar morphological
characteristics, a key to the species of the genus helps to identification of Aphelenchoides species.
Sanwal (1961) produced a dichotomous key to 35 Aphelenchoides species that were recognized at the
time. Shahina (1996) provided a tabular key to 141 Aphelenchoides species and used tail terminus
shape to divide these species into four groups: 1. tail simple without any outgrowth or mucronate
structure; 2. tail terminus with one or sometimes two mucronate structure; 3. tail with tetramucronate
spine or star shape; 4. tail outgrowth other than spine or star. Fortuner (1970) provided a dichotomous
key to 11 Aphelenchoides species with star-shaped mucros. EPPO (2004) devised a polytomous key to
17 Aphelenchoides species including 14 species with star-shaped mucros and 3 species of bud and leaf
nematodes, A. blastophthorus, A. fragariae, A.ritzemabosi without star-shaped mucros, and divided
tail terminus shape of Aphelenchoides species into five groups: 1. tail terminus with star-shaped mucro;
2. tail terminus with a single mucro; 3. tail terminus bifurcate; 4. mucro shape belonging other type at
tail tip; 5. tail terminus without mucro. Allen (1952) provided a key to the 4 species of bud and leaf
nematodes, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis.
[126]
Besides A. besseyi, A. blastophthorus, A. fragariae, A. ritzemabosi and A. subtenuis live as parasites in
bud and leaf of plants, A. saprophilus, a fungivorous species, is also often found in damaged or
diseased plant tissues including bulbs and corms. Andrássy (2007) provided a key to 47
Aphelenchoides species found in European, including the 6 species encountered in buds and leaves. A
short dichotomous key to these bud and leaf nematodes is given in the Table 4 from section 4.2.2.1.
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[127]
Aphelenchoides fragariae is similar to A. arachidis, A. helophilus, A. resinosi, and A. rhytium
(including lateral line unknown species), but can be distinguished from all other species described in
Aphelenchoides by the more slender body (a=45-70), lateral field with two incisures and tail terminus
with a single mucro. A. fragariae can be distinguished from these similar species using the key given
in Table 6. A diagnostic compendium of A. fragariae and similar species, and bud and leaf nematodes
of the genus is presented in Table 7, which provide more details to help to determine the identity of
these similar species.
[128]
Table 6: Dichotomous key to distinguish Aphelenchoides fragariae from morphologically similar species
1
2
female tail length more than 30 µm, conoid to elongate conoid
2
female tail shorter, 22-28 µm long, sub-cylindroid with bluntly rounded tip
A. arachidis
female body length less than 1.0 mm, tail mucro offset; male spicules length less than 25
3
µm
A. helophilus
female body 0.80-1.30 mm, tail mucro no offset; male spicules 26 µm long
3
4
Post-vulval sac length less than half distance between the vulva and the anus
4
Post-vulval sac length more than half distance between the vulva and the anus
A. fragariae
female tail length less than 40 µm, two lateral incisures; spicules 13-15 µm long
A. resinosi
female tail 56.2 µm long, lateral incisures absence; spicules 22.9 µm long
A. rhytium
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2006-025
Table 7: Morphological characters of Aphelenchoides fragariae compared to similar species (adapted from EPPO, 2004)
Species
L(mm)
a
b
c
c’
tail
(µm)
tail shape*
25-42
2-3
22-28
subcylindroid
terminal mucro
shape*
single
central
spine
V
PVS/VA**
stylet
(µm)
L.L.**
spicules
(µm)
References
67-74
approx ½
11-12
2
15-25
Bridge & Hunt,1985
68-70
less than ⅓
10-12
4
18-21
Franklin & Siddiqi,
1972; Andrássy, 2007
Hooper,
1975;
Shahina, 1996
A. arachidis
0.51-1.0
39-50
11-18
A. besseyi
0.66-0.75
32-42
10.2-11.4 17-21
3.5-5.0
36-42
conoid
star
A.blastophthorus
0.68-0.95
28-50
9.0-12.8
15-28
2.3-5.0
42-48
conoid
single
spine
central
62-74
approx ½
15-19.5 4
24-32
A. fragariae
0.45-0.80
36-63
8-15
12-20
4.9
38-42
elongate
conoid
single
spine
central
64-71
more than ½
10-11
2
14-17
A. helophilus
0.80-1.30
43-78
12-14
14-20
5.5
> 40
elongate
conoid
single
spine
central
65-79
12
unknown 26
Shahina,
1996;
Andrássy, 2007
A. resinosi
0.40-0.80
29-53
7-13
12-19
3-4
33.7
conoid
single
spine
central
66-79
less than ½
10-11
2
13-15
Kaisa et al., 1995
A. rhytium
0.78-0.94
43-48
11.7-13.4 16-21
56.2
elongate
conoid
single
spine
central
67
less than ½
11
absent
22.9
Shahina,1996;
Massey, 1974
A.ritzemabosi
0.77-1.20
40-54
10-13
18-24
4-5
47
elongate
conoid
peg with 2-4 66-75
minute processes
more than ½
12
4
20-22
Allen,1952, Siddiqi,
1974; Andrássy, 2007
A. saprophilus
0.45-0.62
26-33
8-12
12-18
2.5-3.0
32
Conoid
a ventral peg
66-70
approx ½
11
4
22-23
Shahina,1996;
Andrássy, 2007
A. subtenuis
0.87-1.15
44-57
12-17
24-28
2.78-3.27 42.4
subcylindroid
single
spine
69-71
more than ½
11
3 or 4
18-23
Allen,1952, Deimi et
al., 2006
ventral
Siddiqi,
Shahina, 1996
1975;
* Shape of female tail and terminal mucro are presented in Figure 13.
** PVS/ VA: Post-vulval sac long / vulva-anus distance; L.L.: number of lateral line.
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Figure 12: Aphelenchoides fragariae : A, N. Female head end; B. Male head end; C. a, female; b,
male, of A. olesistus Ritzema Bos, 1893 [= A. fragariae]; D. a, male; b, posterior portion of female of
Aphelenchus fragariae Ritzema Bos, 1891; E. Male; F. Female; G. Female tail. H, O. Lateral field; I,
M, P. Female tail tip; J, K, Q. Male tails; L. spicules (A, B, E – La after Siddiqi, 1975; C, D after
Ritzema Bos, 1893 and 1891, respectively; M after Allen, 1952; N, Q after Kohl, 2011; O, P after
Khan et al., 2008)
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Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
Figure 13: Tails of Aphelenchoides fragariae and related species of Aphelenchoides. A. A.
arachidis; B. A. besseyi; C. A. blastophthorus; D. A. fragariae; E. A. helophilus; F. A. resinosi; G. A.
rhytium; H. A. ritzemabosi; I. A. saprophilus; J. A. subtenuis ( A after Bridge & Hunt, 1985; B after
Franklin & Siddiqi, 1972; C after Hooper, 1975; D after Allen, 1952; E after Shahina, 1996; F after
Kaisa et al., 1995; G after Massey, 1974; H after Siddiqi, 1974; I after Shahina, 1996; J after Deimi et
al., 2006).
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4.5
2006-025 (5.3)
Morphological identification of A. ritzemabosi
4.5.1 Morphological characteristics of A. ritzemabosi (taken from Siddiqi, 1974)
[132]
Details and views provided in Figure 14.
[133]
Female: Body slender 0.77-1.20 mm long; annules 0.9-1.0 µm wide, distinct; lateral fields 1/6-1/5 as
wide as body, with 4 incisures. Lip region hemispherical set off by a constriction, slightly wider than
adjacent body, no annulations visible with light microscope; frame-work hexaradiate, weakly
sclerotized. Spear about 12 µm long, with distinct basal knobs and sharply pointed anterior part.
Procorpus slender; metacorpus or median oesophageal bulb large, somewhat oval in shape, highly
muscular, with prominent internal cuticular thickening and orifices of dorsal and subventral
oesophageal glands. Nerve ring in neotype 1.5 body widths behind bulb. Excretory pore 0.5-2 body
widths posterior to nerve ring. Three oesophageal glands forming a lobe extending about 4 body
widths over intestine dorsally. Oesophago-intestinal junction abou 8 µm behinf median oesophageal
bulb, indistinct and lacking a valve. Intestine with small spherical granules and a distinct lumen
throughout. Vulva slightly raised, transverse slit. Postvulval uterine sac extending for more than half
the vulva-anus distance, often containing sperms. Ovary single anteriorly outstretched; oocytes in
multiple rows. Tail elongate conoid, bearing a terminal peg which has 2-4 minute processes pointing
posteriorly and giving it a paint-brush like appearance.
[134]
Male: Common. Posterior end of body usually curved through 180 degrees upon relaxation. Lip
region, spear and oesophagus as in female. Testis single, outstretched. Three pairs of
ventro-submedian caudal papillae – first pair adanal, second midway on tail, third near tail end.
Spicules smoothly curved, rosethorn-shaped, lacking a dorsal or ventral process at proximal end;
dorsal limb 20-22 µm long. Tail peg with 2-4 processes, of variable shape.
[135]
Measurements: ♀♀ : L = 077-1.20 mm; a = 40-45; b = 10-13; c = 18-24; V = 48-3366-7514-17. Neotype
♀ : L = 0.85 mm; a = 42; b = 12; c = 18; V = 356817; spear = 12 µm. ♂♂: L = 0.70-0.93 mm; a =
31-50; b = 10-14; c = 16-30; T = 35-64.
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Draft Annex to ISPM 27:2006 – Aphelenchoides besseyi, A. fragariae and A. ritzemabosi
Figure 14: Aphelenchoides ritzemabosi.A: Female head end; B: Female; C: Female tail ends; D: Male
tail ends; E: Female tail; F: Female oesophageal region; G: Spicules; H: Lateral field; I: Male tail
region. (Taken from Siddiqi, 1975)
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4.5.2 Comparison with similar or related species
[137]
Aphelenchoides species are morphologically very similar and can easily be confused. Primary
diagnostic characters are the length of the post-vulval sac (pvs) as a percentage of the distance
between the vulva and the anus, the shape of the tail terminus and tail, body length, and the ratios ‘a’
and ‘c’. More information can be found from Table 3 in section 4.2.1.
4.6
Molecular identification of Aphelenchoides spp.
[138]
Several molecular assays for the identification of Aphelenchoides spp. have been developed and are in
use now. PCR with species specific primers can be used for quicker diagnostics of all development
stages but there is a risk of false negatives if DNA is not extracted properly, so a negative molecular
test result does not exclude the possibility of positive identification by morphological methods.
[139]
For a reliable test result to be obtained the following controls should be considered for each series of
amplification of the target pest or target nucleic acid, depending on the test used and the level of
certainty required:
[140]
-
Positive nucleic acid control: This is used to monitor the efficiency of the test method (apart from
the extraction. Pre-prepared (stored) nematode nucleic acid may be used.
-
Negative amplification control (no template control): This is necessary with conventional to rule
out false positives due to contamination during the preparation of the reaction mix. PCR grade
water that was used to prepare the reaction mix is added at the amplification stage.
-
Interpretation of results from conventional polymerase chain reactions: The pathogen-specific
PCR will only be considered valid if the positive control produces the correct size product for
the target nematode species; and no bands of the correct size for the target nematode species are
produced in the negative amplification control.
DNA may be extracted from single or mixed life stages of nematodes (females, males, juveniles and
eggs). For each assay described below, the DNA isolation methods refer to the source paper for the
original specific DNA extraction technique used. DNA may also be extracted using other extraction
techniques suitable for nematodes. Alternatively, commercial kits for DNA isolation are available.
Additionally, the application amount of PCR was based on the methods of references described in the
following sections, it could also carry on the corresponding reaction according to the instructions of
Taq polymerase enzyme used.
4.6.1 SSU rDNA-based qPCR assay for four foliar nematode species
[141]
Rybarczyk-Mydłowska et al. (2012) designed a small subunit ribosomal DNA (SSU rDNA) -based
species-specific assay against the four foliar nematode species, A. besseyi, A. fragariae, A. ritzemabosi,
and A. subtenuis. The species-specific primers were respectively designed on the base of full-length
SSU rDNA sequences of these four Aphelenchoides species and used for carrying out a quantitative
PCR (qPCR) to rapidly detect plant material and soil for the presence of one or multiple foliar
nematode species.
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Methodology
[142]
[143]
The A. fragariae-specific PCR primers (located in the SSU rDNA sequence) used in this assay were as
follows:
-
1469: 5′-CTT ATC GCA CGA CTT TAC G-3'
-
1472: 5′-TCA AAG TAA TCC GCA TCC AAT-3'
The primer pair (designed with LNA’s) recommended for the detection of A. ritzemabosi is:
-
1496: 5’-CGC TGG TGG GTT TCG A-3’
-
1499: 5’-CCC GCT AAG AAA TGA TCA C*C-3’
* LNA modified nucleotide
[144]
The primer pair (designed with LNA’s) recommended for the detection of A. besseyi is:
1770: 5’-GCG GGA TTC GTG GTT C*T-3’
-
1772: 5’-CGA CAT GCC GAA ACA GAG-3’
* LNA modified nucleotide
[145]
For each quantitative PCR reaction, 3 μl of diluted template was mixed with species-specific primers
(end concentrations for both primers 200 nM) and 12.5 μl of Absolute SYBR Green fluorescein mix
(Thermo Fisher, Wilmington, DE) in a total reaction volume of 25 μl. Thermal cycling was performed
on iQ5 (Bio-Rad, Hercules, CA) and the following PCR profile was used:
[146]
95°C for 15 min; followed by 60 cycles of 95°C for 30 s, 63°C (or gradient from 61 to 66°C) for 1
min, and 72°C for 30 s.
[147]
For negative controls, template was replaced by an equal volume of Milli-Q water. Quantitative PCR
output is expressed in Ct values (cycle number) at which the reporter dye emission intensity crosses a
predetermined threshold.
4.6.2 ITS1 sequence-based PCR assay for A. fragariae
[148]
This assay (McCuiston et al., 2007) was a PCR-based diagnostic assay for early detection and
identification of A. fragariae directly in host plant tissues using the species-specific primers. These
specific primers amplified DNA from A. fragariae and did not amplify DNA from other plant parasitic
nematode species (Meloidogyne incognita, Heterodera schachtii, Pratylenchus penetrans,
Caenorhabditis elegans, Ditylenchus dipsaci, A. besseyi and A. ritzemabosi). The PCR assay was
sensitive, detecting a single nematode in a background of plant tissue extract. The assay had accurately
detected A. fragariae in more than 100 naturally infected, ornamental plant samples including 50 plant
species.
Methodology
[149]
[150]
The PCR primers (within the rDNA-ITS1 region) used in this assay were as follows:
-
Forward primer AFragF1: 5'-GCA AGT GCT ATG CGA TCT TCT-3'
-
Reverse primer AFragR1: 5'-GCC ACA TCG GGT CAT TAT TT-3'
fragariae generated a 169-bp fragment in rDNA-ITS1 region (other species did not amplify DNA)
using the species-specific primers AFragF1 and AFragR1. Reaction mixture is composed of 2.0 μl of
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total DNA extract, 1.25 units of Taq DNA Polymerase , 1×PCR buffer (20 mM Tris-HCl [pH 8.4], 50
mM KCl), 1mM MgCl2, 200 μM of each dNTP, 0.4 μM of each oligonucleotide primer and sterile
molecular grade water to a final volume of 25µL. The cycling conditions are as follows: 94℃ for 2
min, 40 cycles of 94℃ for 1 min, 53℃ for 40 sec and extension at 72℃ for 1 min, followed by a final
extension at 72℃ for 10 min.
4.6.3 18S rRNA gene sequences analysis for A. fragariae and A. ritzemabosi
[151]
This assay of Chizhov et al. (2006) was designed to separate A. fragariae and A. ritzemabosi from
each other and other Aphelenchoididae species (A. blastophthorus, A. bicaudatus, A. besseyi,
Aphelenchoides sp., Bursaphelenchus xylophilus, B. borealis) by using 18S rRNA gene
sequence-based PCR and analyzing the sequences obtained.
Methodology
[152]
The 18S rRNA gene was amplified using the following two sets of primers (sets A+B or sets A+C)
(two overlapping fragments):
-
Set A -forward G18SU: 5'-GCT TGT CTC AAA GAT TAA GCC-3'
-
Set A - reverse R18Tyl1: 5'-GGT CCA AGA ATT TCA CCT CTC-3'
-
Set B - forward F18Ty12: 5'-CAG CCG CGG TAA TTC CAG C-3'
-
Set B - reverse R18Ty12: 5'-CGG TGT GTA CAA AGG GCA GG-3'
or
[153]
-
Set C - forward F18Tyl1: 5'-GTG CCA GCA GCC GCG GTA ATT CC-3'
-
Set C - reverse DF18A-16: 5'-CAC CTA CTC GYA CCT TGT TAC GAC T-3'
The 25 μl reaction mixture is composed as follows: 2.0 μl of extracted DNA, 2.5 μl 10X Taq
incubation buffer, 5 μl Q solution, 0.5 μl dNTPs mixture (Taq PCR Core Kit, Qiagen, Hilden,
Germany), 0.15 μl of each primer (1.0 μg/μl ), 0.2 μl Taq Polymerase and double distilled water to a
final volume. The PCR assay is performed with the following conditions: 94℃ for 4 min, 35 cycles of
94℃ for 1 min, 55℃ for 1.5 min and 72℃ for 2 min, followed by a final extension at 72℃ for 10 min
and quickly cooled to room temperature. The PCR products were analyzed by 0.8% agarose gel
electrophoresis, then cloned and sequenced. The sequence obtained was compared with 18S rRNA
gene sequences of in GenBank by using the ClustalX program.
5． Records
[154]
The records and evidence should be retained as described in section 2.5 of ISPM 27:2006.
[155]
In cases where other contracting parties may be adversely affected by the results of the diagnosis,
especially in these instances of non-compliance and where the nematode is found in an area for the
first time, the records and evidence (in particular following material) should be kept for at least one
year.
[156]
The material required to be kept should include:
-
Original sample of the infested material (labelled appropriately for traceability).
-
Description and photographs of the damage symptoms.
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Measurements and drawings or photographs of the nematode.
-
Permanent slides and / or culture of the nematodes.
-
If relevant, DNA extractions and PCR amplification products should be kept at −80℃ and
−20℃, respectively.
6. Contact Points for Further Information
[157]
USDA, APHIS, PPQ, 389 Oyster Point Blvd., Suite 2, South San Francisco, CA 94080, USA. (Fengru
Zhang, e-mail: [email protected]; Tel.: +1 650 8769098, Fax: +1 650 8760915).
[158]
Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, College of
Natural Resources and Environment, South China Agricultural University, Guangzhou, 510642,
People’s Republic of China. (Hui Xie, e-mail: [email protected]; Tel.: +86 02038297432,
Fax: 86 02038297286).
[159]
Directorate Inspection Services, Department of Agriculture, Forestry and Fisheries, Private Bag
X5015, Stellenbosch 7599, South Africa. (Rinus Knoetze, e-mail: [email protected]. Tel.:
+27 021 8091621)
[160]
Sue Hockland, Independent Plant Nematology Consultant. See www.plantparasiticnematodes.com;
e-mail: [email protected].
[161]
A request for a revision to a diagnostic protocol may be submitted by national plant protection
organizations (NPPOs), regional plant protection organizations (RPPOs) or Commission on
Phytosanitary Measures (CPM) subsidiary bodies through the IPPC Secretariat ([email protected]), which
will in turn forward it to the Technical Panel to develop Diagnostic Protocols (TPDP).
7. Acknowledgements
[162]
This diagnostic protocol was drafted by S. Hockland (author for A. besseyi and A. ritzemabosi), H. Xie
(author for A. fragariae and A. besseyi), R. Knoetze (for A. ritzemabosi and A. fragariae), and F.
Zhang (the lead author for this protocol).
[163]
The information of A. besseyi is adapted from the EPPO Diagnostic Protocol for Aphelenchoides
besseyi (PM7/39(1)), produced by Sue Hockland with the co-operation of the members of the Panel
for Diagnostics in Nematology, the EPPO Secretariat, Dr. David Hunt, and Dr. Mischa Aalten (EPPO,
2004).
[164]
Gratitude is expressed to CAB International for permission to reproduce Figures 8, 10, 11, 14, and part
of 1 and 12, from John Bridge/CABI BioScience (2006) and the CIH Series of Descriptions of
Plant-parasitic Nematodes, Set 1, No.4 (Authors Mary T. Franklin and M.R. Siddiqi, 1972).
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