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A Protocol for Assessing Resistance to Aphelenchoides fragariae in Hosta Cultivars


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Zhen, F., Agudelo, P., and Gerard, P. 2012. A protocol for assessing resistance to Aphelenchoides fragariae in hosta cultivars. Plant Dis. 96:1438-1444. The use of resistant and tolerant cultivars is an important component of an integrated management plan for foliar nematodes on hosta. In order to identify tolerance and resistance in commercial hosta cultivars, reliable and efficient screening methods are required. To optimize the screening protocol, a series of greenhouse experiments was conducted using six hosta cultivars and two types of nematode inoculum. The pathogenicity and reproduction of Aphelenehoides fragariae maintained on fungal cultures versus maintenance on hosta were evaluated with two inoculation methods (with injury and without injury). Both sources of inoculum were pathogenic on all six cultivars tested but the plant inoculum caused two to eight times larger lesions than the fungus inoculum. Both inocula caused larger lesions and resulted in higher reproduction rates on injured leaves than on noninjured leaves. Water soaking was more efficient than traditional Baermann funnel extraction methods. Correlations between foliage symptom severity and nematode reproduction were low or nonexistence. A numerical scale for faster assessment of disease severity was developed, and recommendations for a reliable protocol for assessment of resistance and tolerance are discussed.
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1438 Plant Disease / Vol. 96 No. 10
A Protocol for Assessing Resistance to Aphelenchoides fragariae
in Hosta Cultivars
Fu Zhen and Paula Agudelo, School of Agricultural, Forest, and Environmental Sciences, and Patrick Gerard, Department of Mathe-
matical Sciences, Clemson University, Clemson, SC 29634
Zhen, F., Agudelo, P., and Gerard, P. 2012. A protocol for assessing resistance to Aphelenchoides fragariae in hosta cultivars. Plant Dis. 96:1438-
The use of resistant and tolerant cultivars is an important component of
an integrated management plan for foliar nematodes on hosta. In order
to identify tolerance and resistance in commercial hosta cultivars, relia-
ble and efficient screening methods are required. To optimize the
screening protocol, a series of greenhouse experiments was conducted
using six hosta cultivars and two types of nematode inoculum. The
pathogenicity and reproduction of Aphelenchoides fragariae main-
tained on fungal cultures versus maintenance on hosta were evaluated
with two inoculation methods (with injury and without injury). Both
sources of inoculum were pathogenic on all six cultivars tested but the
plant inoculum caused two to eight times larger lesions than the fungus
inoculum. Both inocula caused larger lesions and resulted in higher
reproduction rates on injured leaves than on noninjured leaves. Water
soaking was more efficient than traditional Baermann funnel extraction
methods. Correlations between foliage symptom severity and nematode
reproduction were low or nonexistence. A numerical scale for faster
assessment of disease severity was developed, and recommendations for
a reliable protocol for assessment of resistance and tolerance are
Foliar nematodes Aphelenchoides fragariae (Ritzema Bos,
1890) Christie, 1932 (Aphelenchida: Aphlenchidae) are endo- and
ectoparasites of many plants, including ornamental and agricultural
crops (3,21). In nurseries and landscapes in the United States, these
nematodes can be a serious problem (11–13) affecting hosta (Hosta
spp.), a commonly grown herbaceous ornamental that thrives in
shady environments. The nematodes can enter the foliar tissue
through stomata or wounds (13,23) and feed on mesophyll cells
(19,23), causing characteristic vein-delimited lesions that start as
lightly chlorotic and then turn necrotic. The nematodes may over-
winter in the soil, dormant crowns, and dry leaves (13). In temper-
ate regions, they migrate to the new leaves in the spring (13).
Control of foliar nematodes on hosta can be difficult because of
the survival behaviors of the nematode (3,13) and because hosta
cultivars are popular perennial plants adapted to a wide geograph-
ical range, with numerous species and cultivars grown (20). Be-
cause thousands of plants are traded each year, there is increasing
concern among growers about the movement of this nematode and
fear of dissemination to noninfested areas. Forty-seven countries
have legislation regulating the movement of this species in interna-
tional trade (15). In order to develop adequate management strate-
gies, it is important to effectively combine the knowledge of culti-
var susceptibility with the existing chemical and cultural control
options (11–14). However, there is currently no standardized
method for assessing resistance of hosta cultivars to foliar nema-
todes. Jagdale and Grewal (13) tested the pathogenicity of A.
fragariae on 23 cultivars, and found it pathogenic on 20 of them.
They measured presence or absence of symptoms but did not as-
sess symptom severity or nematode reproduction. Because the
value of hosta plants lies mainly in the quality of their foliage, it is
important to evaluate symptom severity. Symptom expression,
however, can be highly variable due to influence of the environ-
ment and characteristics of the foliage of each cultivar. When
symptom development is slow, asymptomatic plants may support
nematode reproduction (17). Consequently, nematode reproduction
becomes an important part in the assessment of plant resistance
and should be measured along with symptom severity. Breeders
and growers would benefit from a standard protocol that assesses
plant resistance and nematode reproduction (3).
The objective of this study was to develop a standardized proto-
col for the assessment of resistance to A. fragariae on hosta culti-
vars. In order to select the best procedures, we evaluated the effects
of inoculum type (maintained on cultured fungus versus main-
tained on plants), inoculation method (with injury versus without
injury), and nematode harvesting methods. We also explored po-
tential correlations between nematode reproduction and symptom
Materials and Methods
Hosta plants. Certified nematode-free hosta plants of six culti-
vars, representing a diversity of Hosta spp. commonly cultivated in
the United States, were obtained from a commercial nursery in
Spartanburg, SC. The cultivars selected included ‘Albo Marginata’
(Hosta sieboldii), ‘Aureo Marginata’ (H. ventricosa), ‘Fragrant
Bouquet’ (H. plantaginea), ‘Golden Tiara’ (H. nakaiana), ‘Guaca-
mole’ (H. plantaginea), and ‘Patriot’ (H. sieboldiana). Henceforth,
we will refer to them by cultivar name only. The plants were grown
in the greenhouse in individual pots until they had at least eight
leaves. At least 30 plants of each cultivar were used to conduct
these studies, according to the treatments described below.
Nematode inoculum sources. The effect of two types of A.
fragariae inoculum (one maintained on a fungal culture [“fungus”]
and one maintained on hosta plants [“plant”]) on pathogenicity,
symptom severity, and host suitability on the six hosta cultivars
was evaluated. The fungus nematodes are part of the Clemson Uni-
versity Nematode Collection and have been cultured in vitro on
Cylindrocladium sp. grown in potato dextrose agar (HiMedia
Laboratories) under laboratory conditions for 25 years. The origin
of the isolate is uncertain. The plant nematodes were isolated from
infected hosta in the South Carolina Botanical Garden, identified
by morphology, and cultured on hosta in the field and the green-
house. To obtain the fungus inoculum, nematodes were extracted
by Baermann funnel (2) and a water suspension of mixed develop-
mental stages from the extraction was adjusted to a concentration
Corresponding author: P. Agudelo, E-mail:
Accepted for publication 6 May 2012. / PDIS-10-11-0895-RE
2012 The American Phytopathological Society
Plant Disease / October 2012 1439
of 5,000 individuals per milliliter before inoculation. To obtain the
plant inoculum, hosta leaves infected with A. fragariae were cut
into 1-cm2 pieces and soaked in water for 24 h. The mix was
poured through nested sieves of 20 mesh (850 µm) and 500 mesh
(25 µm) and the contents of the 500-mesh sieve were transferred to
a Baermann funnel. The extract was washed several times with
sterilized tap water and adjusted to the same concentration as the
fungus inoculum. Each inoculum type was evaluated with each
inoculation method (described below) on all six cultivars.
Leaf inoculation methods. Two inoculation methods (with and
without leaf injury) were evaluated. Each combination of inoculum
type and inoculation method was replicated on five plants of each
of the six cultivars. In all treatments, nematode inoculation was
conducted on two arbitrarily selected leaves of each plant. One leaf
was not injured prior to inoculation and the other was injured by
one of two methods: with a scalpel, by making five short cuts in
the upper side of the leaf, or with a needle, by making 10 perfora-
tions scattered between the leaf veins. Both leaves were wrapped
with wet tissue paper (Kimwipes, 11 by 21 cm; Kimberly-Clark)
and 1 ml of the suspension of the nematodes was carefully dis-
pensed on the tissue paper. The plants were covered with black
plastic bags after inoculation in order to maintain a moist
environment. The bags and tissue wrapping were removed after 72
h. All plants were kept in a shaded greenhouse at 25 ± 5°C.
Treatments were arranged in a randomized complete block design
and the experiment was performed twice.
Data collection. Inoculated hosta leaves were harvested 35 days
after inoculation. Symptom severity was evaluated by calculating
the percentage of affected leaf area using the grid method. Photo-
graphs of each inoculated leaf were taken and later used to aid in
the development of a rating key. The leaves were cut into 1-cm2
pieces and soaked in tap water for 48 h at room temperature. The
nematodes that emerged from the leaf pieces were recovered using
nested sieves of 20 mesh (850 µm) and 500 mesh (25 µm) and
Comparison of efficiency of extraction methods. Three har-
vesting methods were evaluated: traditional Baermann funnel,
modified Baermann funnel, and water soaking. Symptomatic hosta
leaves from different cultivars were collected, cut into 1-cm2
pieces, and mixed together. Leaf pieces were equally divided
among treatments and weighed before processing. The traditional
Baermann funnel method was evaluated at room temperature (22 ±
2°C), whereas the modified Baermann funnel and water-soaking
methods were evaluated at both room temperature and 28 ± 1°C.
Three replicates were included for each treatment. The amount of
nematodes recovered after 24 h was counted and recorded. For the
traditional Baermann funnel method, leaf pieces were wrapped
with tissue paper (Kimwipes, 11 by 21 cm) and placed in a glass
funnel filled with tap water. For the modified Baermann funnel
method, leaf pieces were wrapped with large Kimwipes (37 by 42
cm) and placed on a 20-mesh sieve (25 cm in diameter, 850-µm
openings). The sieve was placed in a plastic container with tap
water just covering the leaf material. A small aquarium pump was
used to aerate the water during the incubation. For the water-
soaking method, leaf pieces were placed in petri dishes (10 cm in
diameter) filled with tap water. The nematodes that emerged from
the leaf pieces were recovered using nested sieves of 20 mesh (850
µm) and 500 mesh (25 µm) and counted.
Statistical analysis. All nematode density data were natural log-
transformed, except for those for the extraction method experi-
ment. Nematode density, symptom severity, and extraction effi-
ciency data were analyzed by one-way analysis of variance with
Fig. 1. Effect of Aphelenchoides fragariae inoculum type (maintained on fungus or plants) on nematode density on six hosta cultivars: ‘Albo Marginata’, ‘Fragrant Bouquet’,
‘Golden Tiara’, ‘Guacamole’, ‘Patriot’, and ‘Aureo Marginata’. Error bars are standard error of the mean (n = 20). Different letters indicate significant (P < 0.05) differences
within the same cultivars according to Student’s t test.
Tab l e 1 . Severity of symptoms, measured as percentage of leaf area with lesions, caused by Aphelenchoides fragariae maintained on hosta (plant inoculum)
versus Cylindrocladium spp. (fungus inoculum) on four hosta cultivars (n = 10)
Leaf area with lesions (%)z
Type of inoculum Albo Marginata Aureo Marginata Guacamole Patriot
Plant inoculum 10.34 ± 1.79 A 4.17 ± 1.51 A 25.95 ± 0.85 A 10.34 ± 3.43 A
Fungus inoculum 4.13 ± 0.75 B 1.21 ± 0.67 B 3.53 ± 0.71 B 1.22 ± 0.55 B
z Different letters indicate significant differences (P < 0.05) within the same column (cultivar) according to Student’s t tests.
1440 Plant Disease / Vol. 96 No. 10
JMP 9 software (SAS Institute). Differences between treatments
were determined by Fisher’s least significant difference or Stu-
dent’s t test at P < 0.05. Potential correlations between symptom
severity and nematode density were analyzed using the Bivariate fit
procedure of JMP 9 software. If there were no differences between
repeated experiments (P > 0.05), data were combined for analysis.
There was no effect of the separate trials (P > 0.05); therefore, the
results for each experiment are presented based on combined trials.
Effect of inoculum source. Both types of inoculum (plant and
fungus) were pathogenic to all six cultivars tested but there were
Fig. 3. Comparison of five extraction methods for recovery of Aphelenchoides fragariae individuals from hosta (Hosta spp.) leaves. Error bars are standard errors of the
means. Extraction methods not sharing a common letter indicate significant (P < 0.05) differences according to Fisher’s least significant difference (P < 0.05).
Fig. 2. Effect of injury on Aphelenchoides fragariae density following application of two types of inoculum (maintained on fungus or plants). Error bars are standard error o
the mean (n = 44). Different letters indicate significant (P < 0.05) differences within the same inoculum type according to Student’s t test.
Plant Disease / October 2012 1441
significant differences (P < 0.05) between the two types of inocu-
lum sources in nematode reproduction and disease severity (Fig. 1;
Table 1). Disease severity, measured as percentage of the leaf area
with lesions, was higher in leaves treated with the plant inoculum
(Table 1). Characteristic, vein-delimited lesions were observed as
early as 21 days after inoculation and progressed from yellow to
brown in color as time passed. Overall, lesions were two to eight
times larger with plant inoculum than with fungus inoculum. The
largest differences in disease severity as a result of inoculum type
were observed with Patriot, where lesions were about 10% of leaf
area with plant inoculum and about 1% with fungus inoculum. The
disease severity data for Fragrant Bouquet and Golden Tiara were
not included in Table 1 because the occurrence of other foliar dis-
ease symptoms compromised the reliability of the symptom sever-
ity evaluation. However, we did include the nematode density data
for all six cultivars. On five of the six cultivars, nematode repro-
duction was higher in the leaves inoculated with plant inoculum
(Fig. 1). Only Albo Marginata supported higher nematode densities
per leaf with the fungus inoculum (Fig. 1). There were differences
(P < 0.05) between nematode densities obtained with plant inocu-
lum and with fungus inoculum on Albo Marginata, Golden Tiara,
Guacamole, and Patriot. For Fragrant Bouquet and Aureo Mar-
ginata, there were no statistical differences between the plant and
fungus inocula. The highest nematode reproduction was observed
on Aureo Marginata (17,400 individuals/leaf recovered 35 days
after inoculation), using plant inoculum.
Effect of inoculation method. There were differences (P <
0.05) in nematode reproduction and disease severity between
inoculation treatments with and without injury (Fig. 2; Table 2).
Symptom severity of leaves injured at the time of inoculation was
greater when observed 35 days after inoculation. The difference in
disease severity was more noticeable with plant inoculum than
with fungus inoculum. The combined mean size of the lesions was
doubled for fungus inoculum when aided by injury but was more
than eight times larger for plant inoculum when aided by injury.
Nematode reproduction was enhanced in a similar manner (Table
2). When combining the data for all cultivars, nematode densities
were up to 38-fold higher on injured leaves with plant inoculum,
and up to 3.7-fold higher on injured leaves with fungus inoculum.
Comparison of efficiency of extraction methods. Numbers of
nematodes extracted from the same weight of leaf material were
different (P < 0.05), depending on the extraction method used (Fig.
3). The traditional Baermann funnel technique yielded the lowest
number of individuals but it was the most consistent technique
(lowest standard deviation), and the extracts also contained the
least leaf debris. This is important because it makes the nematodes
within the extracts easier to quantify. The most efficient extraction
method tested was water soaking, which yielded more than four
times more nematodes than were obtained than with the traditional
Baermann funnel technique. Incubation temperature did not change
the efficiency of the water-soaking or the modified Baermann fun-
nel techniques. The modified Baermann funnel technique yielded
extracts with the most debris and, consequently, was the most diffi-
cult to quantify.
Correlation between disease severity and nematode density.
For three of four cultivars, the severity of symptoms increased
directly with nematode population but the level of correlation var-
ied by cultivar (Fig. 4). For Albo Marginata, nematode population
density and disease severity were not correlated. A low positive
correlation (r = 0.538; P < 0.05) between these two variables was
observed for Guacamole. Higher positive correlations were ob-
served with Aureo Marginata (r = 0.734; P < 0.05) and Patriot (r =
0.856; P < 0.05). Equations were derived to describe relationships
between population density of A. fragariae and symptom severity
for the three cultivars with positive correlations (Fig. 4). In each,
the slope of the line is a measure of the susceptibility of the culti-
var. The most susceptible cultivar (i.e., the one with the largest
lesions caused by a given number of nematodes) was Guacamole.
Fig. 4. Regression analysis of symptom severity (expressed as percentage of lea
area with lesions) caused by Aphelenchoides fragariae versus nematode density on
three Hosta cultivars: A, ‘Aureo Marginata’; B, ‘Guacamole’; and C, ‘Patriot’.
Tab l e 2. Severity of symptoms, measured as percentage of leaf area with
lesions, caused by Aphelenchoides fragariae maintained on hosta (plan
inoculum) versus Cylindrocladium spp. (fungus inoculum) and inoculated
on leaves with and without mechanical injuryy
Leaf area with lesions (%)z
Inoculation method Plant inoculum Fungus inoculum
Injured 22.53 ± 5.76 A 3.23 ± 0.52 A
Noninjured 2.68 ± 0.76 B 1.60 ± 0.47 B
yData for six cultivars were combined (n = 30). Injuries were made with a
needle (10 perforations per leaf).
zDifferent letters indicate significant differences (P < 0.05) within the
same column (inoculum type) according to Student’s t tests.
1442 Plant Disease / Vol. 96 No. 10
The use of resistant and tolerant cultivars is an important
component of an integrated management plan for foliar nematodes
on hosta. In order to identify tolerance and resistance in commer-
cial hosta cultivars, reliable and efficient screening methods are
required. Based on the results of the experiments of this study and
on our experience working with this nematode, we recommend the
following protocol for screening for resistance to foliar nematode
on hosta cultivars. Maintain and increase foliar nematode inoculum
on hosta plants in the greenhouse. We recommend Patriot and Gua-
camole for this purpose, because they are commonly grown culti-
vars that sustain adequate nematode reproduction. For inoculum
extraction, cut infected hosta leaves into 1-cm2 pieces and soak in
tap water for 48 h at room temperature. Pour the mix through
nested sieves of 20 mesh (850 µm) and 500 mesh (25 µm). Wash
contents collected on the 500-mesh sieve with tap water. Adjust the
concentration of the extract to 5,000 mixed stage individuals per
milliliter and use within 3 days of extraction. Hosta plants to be
evaluated should be potted individually, and each plant to be inocu-
lated should have at least eight leaves. Select two healthy leaves on
each hosta plant and make 10 perforations, scattered between leaf
veins, with a needle on both leaves. Wrap both leaves with wet
tissue paper (e.g., Kimwipes, 11 by 21 cm). Dispense 1 ml of the
suspension of nematodes on one leaf, and 1 ml of sterile tap water
on the other leaf as negative control. Cover the plants with black
plastic bags. After 72 h, remove the bags and tissue wrappings. Use
a completely randomized arrangement for the experimental design
with at least five replications. Run the experiment at least twice.
One susceptible cultivar should be included in each experiment as
positive control. Based on the results of this study, we recommend
Guacamole as the susceptible control cultivar. Maintain the green-
house at 25 ± 5°C and use shade cloth (50 to 60% density). Water
the plants carefully to avoid splashing. Provide a light-and-dark
phase of 12 h, supplementing with artificial lights when necessary.
After 35 days post inoculation, collect inoculated leaves and as-
sess symptom severity of each, using a 0-to-6 scale (Figs. 5 and 6)
based on the percentage of the total leaf area with lesions and
chlorosis. Evaluate the positive and negative controls first. If the
positive control is rated 0 or 1, discard the test and run again. If the
negative control (i.e., the leaves inoculated with water) is not rated
0, discard the test and run again. Cut inoculated leaves into 1-cm2
pieces and soak in a 10-cm-diameter glass petri plate with 40 ml of
tap water for 48 h at room temperature. Retrieve the nematodes
passing through nested sieves of 20 mesh (850 µm) and 500 mesh
(25 µm) and count. Report the numerical rating for symptom
Fig. 5. Rating chart of symptom severity on hosta caused by foliar nematode (Aphelenchoides fragariae). Drawings of hosta leaves are based on appearance of ‘Guacamole’ hosta.
Plant Disease / October 2012 1443
severity and the nematode density data for each plant and
Variability in rates of reproduction of A. fragariae in experi-
mental culture is often high (3). There have been very few
studies on the fate of nematodes immediately after inoculation
but loss is believed to be very high. Plowright and Gill (18)
estimated that more than 75% of Ditylenchus angustus inoc-
ulum was lost after inoculation. Our observations support the
use of wet tissue and plastic bags for minimizing inoculum
loss, although we have simply observed improved infection and
have not quantified losses. These observations are also sup-
ported by the fact that a common cultural control recom-
mendation for foliar nematodes is to avoid excess surface mois-
ture of the foliage (21).
It is convenient to rear nematodes in pure cultures either in fun-
gal cultures or on callus tissue. Several migratory endoparasitic
nematodes such as Pratylenchus spp., Radopholus spp., and
Ditylenchus spp. are routinely cultured on carrot disks or using
other in vitro methods. Some authors report changes in infectivity
of the inoculum reared in vitro (22) while others report no differ-
ences (5,10). Ali and Ishibashi (1) recommend that infectivity and
aggressiveness of Ditylenchus spp. reared in monoxenic culture be
monitored on the field host. In contrast, Erikson (6) reports having
successfully maintained and regularly subcultured a lucerne race of
D. dipsaci on callus for more than 10 years.
In our studies, A. fragariae lost virulence when maintained on
fungal cultures. Researchers who, for convenience, decide to use
inoculum reared on fungi should be aware that symptom expres-
sion and nematode reproduction can be greatly reduced. Addition-
ally, culturing nematodes on fungi comes with the risk of inoculat-
ing plants with fungal propagules along with the nematode
inoculum (16). This is an important risk if the fungal host is patho-
Fig. 6. Hosta (Hosta spp.) leaves illustrating the different degrees of foliar nematode (Aphelenchoides fragariae) symptom severity, according to our suggested rating chart.
A, Leaf of ‘Guacamole’, rated 0. B, Leaf of ‘Golden Tiara’, rated 1. C, Leaf of ‘Patriot’, rated 2. D, Leaf of ‘Green Piecrust’, rated 3. E, Leaf of Guacamole, rated 4. F, Leaf o
Guacamole, rated 5.
1444 Plant Disease / Vol. 96 No. 10
genic to plants; for example, Rhizoctonia solani (13), Botrytis ci-
nerea (3), and Cylindrocladium sp. (this study).
Wingfield (24) observed that races of Bursaphelenchus xy-
lophilus with low pathogenicity to pine could be more vigorous in
the mycophagous phase. A. fragariae is closely related to B. xy-
lophilus and similar to it in its ability to feed on both fungi and
plants (3,13,21); however, distinct phytophagous and mycophagous
phases of the foliar nematode life cycle have not been character-
ized. Fungal feeding in foliar nematodes is believed to play an
important role in survival in the soil and plant debris, and it is
possible that isolates with relatively low virulence are more vigor-
ous when feeding on fungi than when feeding on plants. This hy-
pothesis is yet to be tested.
We recommend injuring hosta leaves with a needle as part of the
inoculation procedure. Our research showed that foliar nematodes
will infect hosta plants without the aid of injury but that certain
cultivars will show few to no symptoms without injury. Jagdale and
Grewal (13) also demonstrated that injury was necessary to cause
symptoms on cultivars such as ‘Fried Green Tomatoes’ and ‘Fra-
grant’. In our study, we found the same was true for Patriot. We
recommend small perforations (>1 mm) made with a sharp needle.
In the control mock inoculations with water, these perforations
were healed by 35 days. For cultivars with more succulent leaves,
injury with a scalpel (cuts) should be avoided, because we ob-
served that these are readily colonized by opportunistic fungi. Be-
cause injury cannot be completely avoided in commercial settings
(transporting, cultural practices, and so on) or in nature (wind,
insects, and so on), we believe that injury should be part of the
standard protocol.
We recommend the water-soaking method at room temperature
for extraction of foliar nematodes from leaf tissue. This method is
simple and yielded adequate amounts of nematodes free from leaf
tissue debris. The traditional Baermann funnel technique yielded a
cleaner extract and also required little labor and simple equipment
but the extraction efficiency was lower. Fortuner (7) reported that
specimens can be trapped by the tissue and the sides of the funnel
with this extraction method. We were expecting the extraction
efficiency to be improved by aeration (modified Baermann), be-
cause oxygenation of the water has been reported to improve
migration of nematodes from the plant tissue (8,9), but we did not
observe such improvement. It is possible that aerating favored the
growth of fungi and bacteria that could have affected nematode
Differences in host response and nematode reproduction be-
tween series of experiments conducted under similar conditions
are not uncommon in nematology (4). Such differences may arise
from differences in the environmental conditions, developmental
stage of plants, and infectivity of the inoculum. The occurrence
of these differences underlines the importance of including the
same susceptible control host in each experiment. This way,
comparison of results obtained from different tests is possible.
The variability of correlation between symptom expression and
nematode reproduction in our studies indicates the importance of
measuring both parameters when evaluating cultivars. Our sug-
gested protocol assesses both resistance (reduced nematode
reproduction) and tolerance (reduced loss of aesthetic value
caused by symptom expression, regardless of nematode repro-
duction), and provides a useful tool for breeders, nematologists,
consultants, and extension specialists in the development of
new cultivars and of recommendations regarding cultivar selec-
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... We also describe the population dynamics of the nematode in different types of tissues as they fluctuate throughout the year as a contribution to our understanding of BLD development and the life history of Litylenchus nematodes, with the goal of improving BLD surveys. In addition, we assessed if a simple, modified pan method could be used to detect nematodes in different types of tissues at different times of the year (Townshend, 1963;Zhen, Agudelo, & Gerard, 2012 In Ontario, three forested locations with BLD were identified in June 2018. These locations were within 46 km of one another and within 9 km of Lake Erie near Alymer, Port Stanley, and Welsingham in Elgin and Norfolk counties. ...
... Nematodes were extracted using a quick, easy, modified pan method, also called a water-soaking method, as these are quick, easy, and can be carried out in laboratories without equipment for DNAbased assays (Townshend, 1963;Zhen et al., 2012). In Ontario, three leaves were randomly selected from each sample. ...
... In Ohio, the entire sample of five leaves was cut into 2 cm 2 pieces before soaking overnight at 22°C (Zhen et al., 2012). The sample was centrifuged at 1252 g for 3 min to form a nematode pellet. ...
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A foliar nematode, Litylenchus crenatae ssp. mccannii, is associated with beech leaf disease (BLD) symptoms. Information about the types of tissues parasitized and how nematode populations fluctuate in these tissues over time is needed to improve surveys as well as understand the nematodes role in BLD. During this study, the nematode was detected throughout the known range of BLD by researchers at both Canadian and US institutions using a modified pan method to extract nematodes. Monthly collections of symptomatic and asymptomatic leaves during the growing season (May–October), and leaves and buds between growing seasons (November–March), revealed that nematodes were present in all tissue types. Progressively larger numbers of nematodes were detected in symptomatic leaves from Ohio and Ontario, with the greatest detections at the end of the growing season. Smaller numbers of nematodes were detected in asymptomatic leaves from BLD‐infected trees, typically at the end of the growing season. The nematode was detected overwintering in buds and detached leaves. The discovery of small numbers of nematodes in detached leaves at one location before BLD was detected indicates that nematodes may have been present before disease symptoms were expressed. Other nematodes, Plectus and Aphelenchoides spp., were infrequently detected in small numbers. Our findings support the involvement of the nematode in BLD and indicate that symptoms develop only when certain requirements, such as infection of buds, are met. We also found that the nematode can be reliably detected in buds and leaves using the modified pan extraction method.
... Nematodes were isolated from leaves collected with severe BLD symptoms, and a modification of the "water soaking" isolation method (Zhen, Agudelo, & Gerard, 2012) was employed. In short, 5 leaves with symptoms of BLD were cut into 1-cm 2 pieces and placed in a petri dish containing 4% potato dextrose agar. ...
... Leaves were injured using a sterile dissecting needle by making small holes in the leaf tissue and by scraping the needle across the underside and upper side of each leaf. Leaves were injured as this was found to produce the highest level of nematode leaf colonization in previous work (Zhen et al., 2012). After leaf injury, the leaf was wrapped in a 11 × 21 cm Kimwipe (Kimberly-Clark) which was lightly moistened with sterile water to make it adhere to the leaf surface. ...
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Symptoms of beech leaf disease (BLD), first reported in Ohio in 2012, include interveinal greening, thickening and often chlorosis in leaves, canopy thinning and mortality. Nematodes from diseased leaves of American beech (Fagus grandifolia) sent by the Ohio Department of Agriculture to the USDA, Beltsville, MD in autumn 2017 were identified as the first recorded North American population of Litylenchus crenatae (Nematology, 21, 2019, 5), originally described from Japan. This and other populations from Ohio, Pennsylvania and the neighbouring province of Ontario, Canada showed some differences in morphometric averages among females compared to the Japanese population. Ribosomal DNA marker sequences were nearly identical to the population from Japan. A sequence for the COI marker was also generated, although it was not available from the Japanese population. The nematode was not encountered in Fagus crenata (its host in Japan) living among nematode‐infested Fagus grandifolia in the Holden Arboretum, nor has L. crenatae been reported in American beech in Japan. The morphological and host range differences in North American populations are nomenclaturally distinguished as L. crenatae mccannii ssp. n. from the population in Japan. Low‐temperature scanning electron microscopy (LT‐SEM) demonstrated five lip annules and a highly flexible cuticle. Females, juveniles and eggs were imaged within buds with a Hirox Digital microscope and an LT‐SEM. Nematodes swarmed to the tips of freshly cut beech buds, but explants could not be maintained. Inoculation of fresh nematodes from infested leaves or buds to buds or leaves of F. grandifolia seedlings resulted in BLD leaf symptoms. Injuring dormant buds prior to nematode application, in fall or spring, promoted the most reliable symptom expression. The biogeography and physiology of anguinid nematode leaf galling, and potential co‐factors and transmission are discussed.
... The minimum number of Ab-XI nematodes was 1329.33 when inoculated in Sha ecotype, which was significantly lower (P < 0.05) than that in other ecotypes. The number 24 , the rating of symptom severity on A. thaliana caused by foliar nematodes were assigned as follows: rated 0 = no lesion/chlorosis, 1 = 10% lesion/chlorosis, 2 = 11-25% lesion/chlorosis, 3 = 26-50% lesion/chlorosis, 4 = 51-75% lesion/chlorosis, 5 = 75% or more lesion/chlorosis. Ab-S24 and Ab-XI were A. besseyi, CFN was A. ritzemabosi. of nematodes was not significantly different (P > 0.05) among Chi, Ler and Ws ecotypes. ...
... Symptom observations. Acording to Zhen et al. 24 , the rating of symptom severity on A. thaliana caused by foliar nematodes were assigned as follows: rated 0 = no lesion/chlorosis, 1 = 10% lesion/chlorosis, 2 = 11-25% lesion/chlorosis, 3 = 26-50% lesion/chlorosis, 4 = 51-75% lesion/chlorosis, 5 = 75% or more lesion/chlorosis. Symptomatic leaves were selected at random and stained with acid fuchsin according to Volvas et al. 25 . ...
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The rice white tip nematode (RWTN), Aphelenchoides besseyi and the chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi are migratory plant parasitic nematodes that infect the aboveground parts of plants. In this research, Arabidopsis thaliana was infected by RWTN and CFN under indoor aseptic cultivation, and the nematodes caused recognizable symptoms in the leaves. Furthermore, RWTN and CFN completed their life cycles and proliferated. Therefore, A. thaliana was identified as a new host of RWTN and CFN. The optimum inoculum concentration for RWTN and CFN was 100 nematodes/plantlet, and the optimum inoculum times were 21 and 24 days, respectively. For different RWTN populations, the pathogenicity and reproduction rates were different in the A. thaliana Col-0 ecotype and were positively correlated. The optimum A. thaliana ecotypes were Col-0 and WS, which were the most susceptible to RWTN and CFN, respectively. Additionally, RWTN was ectoparasitic and CFN was ecto- and endoparasitic in A. thaliana. The RWTN and CFN migrated from inoculated leaves to the entire plantlet, and the number of nematodes in different parts of A. thaliana was not correlated with distance from the inoculum point. This is a detailed study of the behavior and infection process of foliar nematodes on A. thaliana.
... Some species of plants are genetically resistant to certain PPNs, such as resistance to some Aphelenchoides spp. on hosta, but the extent of plant resistance is largely unknown and focuses more on agricultural crops. Protocols to assess resistance have been established in certain plant species, such as on Aphelenchoides spp. in hosta (Zhen et al., 2012), but this is not available in most ornamental plants. An important aspect of all pathogen management is integrated pest management (IPM), which is a cornerstone of ornamental plant and nursery crop production (Daughtrey and Benson, 2005). ...
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Worldwide, the ornamental plant industry is estimated to be valued at $70 billion, with the United States’ ornamental plant industry valued at $4.8 billion in 2020. Ornamental plants are cultivated for numerous reasons worldwide, such as decorative, medicinal, social, and utility purposes, making the ornamental field a high growth industry. One of the main pathogen groups affecting the yield and growth of the ornamental plant industry is plant-parasitic nematodes, which are microscopic roundworms that feed on plant parts causing significant yield loss. There are many kinds of plant-parasitic nematodes that affect ornamental plants, with the main genera being Meloidogyne spp., Aphelenchoides spp., Paratylenchus spp., Pratylenchus spp., Helicotylenchus spp., Radopholus spp., Xiphinema spp., Trichodorus spp., Paratrichodorus spp. , Rotylenchulus spp., and Longidorus spp. The aim of this review is to focus on the effects, hosts, and symptoms of these major plant-parasitic nematodes on ornamental plants and synthesize current management strategies in the ornamental plant industry.
... This issue improved by farmers, but nematode spread is still high. Generally, cultural management programs should include the removal and destruction of infected plants and debris, abscised leaves in pots/ground should be disposed, in addition, sterilizing the pots and equipments (trowel, pruning shears/pruning saw, scissors), avoid sprinkler irrigation, and misting which can create an ideal condition for nematode dispersal (Young, 2000;Zhen et al., 2012). The use of certified nematode-free planting materials can prevent the spread of plant parasitic nematodes (PPN), such as Aphelenchoides besseyi on hosts (Coyne et al., 2013). ...
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Nematodes are hidden enemies inhibiting the entire ecosystem causing adverse effects on animals and plants, leading to economic losses. Management of foliar phytoparasitic nematodes is an excruciating task. Several techniques were used such as traditional practices, resistant cultivars, plant extract, compost, biofumigants, induced resistance, nano-biotechnology applications, and chemical control. This study reviews the various strategies adopted in combating plant-parasitic nematodes while examining the benefits and challenges. The significant awareness of biological and environmental factors determines the effectiveness of nematode control, where the incorporation of alternative methods to reduce the nematodes population in plants with increasing crop yield. The researchers were interested in explaining the basic molecular mechanisms, providing an opportunity to deepen our understanding of the sustainable management of nematodes in croplands. Eco-friendly pesticides are effective as a sustainable nematodes management tool and safe for humans. The current review presents the eco-friendly methods in controlling nematodes to minimize yield losses, and benefit the agricultural production efficiency and the environment.
... In brief, we used the "water soaking" method of Zhen, Agudelo, and Gerard (2012) to isolate nematodes as it was found to produce the largest number of nematodes. Leaves were collected from the lower Baldwin site in July 2018, cut into 1-cm 2 pieces and soaked overnight in sterile water for 24 hr at 22°C. ...
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American beech (Fagus grandifolia) is the target of a newly emerging disease in North America called beech leaf disease (BLD) that affects and disfigures leaves and which can lead to tree mortality. Beech leaf disease may be caused by a newly recognized subspecies of the anguinid nematode Litylenchus crenatae subsp. mccannii, but the associations of this nematode with bacterial and fungal taxa are unknown. We examined microbial communities associated with beech leaves affected by BLD in a 16‐year‐old American beech plantation using molecular methods. We detected L. crenatae subsp. mccannii in anywhere from 45% to 90% of leaves depending on the degree of visual BLD symptoms. Approximately 37% of asymptomatic leaves contained L. crenatae subsp. mccannii, whereas 90% of buds associated with symptomatic leaves contained L. crenatae subsp. mccannii. We found that fungal communities on leaves and buds were unaffected by BLD, but bud and leaves had significantly different fungal communities. Bacterial communities on buds also were unaffected by BLD, but bacterial communities were significantly different between symptomatic and asymptomatic leaves suggesting that the nematode could be altering the community of bacteria on the leaves. Clone libraries indicate that Wolbachia, an intracellular endosymbiont of arthropods, was found only on symptomatic leaves and buds associated with either symptomatic or asymptomatic leaves. In addition, only symptomatic leaves contained taxa in the genus Mucilaginibacter, which previous studies suggest could produce exopolysaccharides. These bacterial taxa could represent a marker for the vector of L. crenatae subsp. mccannii that enables spread between trees and a possible endosymbiont that could facilitate nematode feeding and establishment on nematode infested leaves. Our results are the first to examine changes to the leaf microbiome of this newly emerging pest and may aid identification of mechanisms associated with the spread and success of L. crenatae subsp. mccanni.
... Isolates of B. xylophilus that are more vigorous in the mycophagous phase have shown reduced virulence on pine (26). Likewise, we have observed differences in virulence between A. fragariae isolates reared on fungi versus plants (3). The mechanisms associated with feeding preferences and the factors that govern the coordination of the mycophagous and phytophagous behaviors in the life cycle of foliar nematodes have not been elucidated. ...
... Gardeners grow hostas because of their attractive leaf shapes, texture and color, and low maintenance costs. However, there is a growing concern among the growers and nursery managers about the serious leaf damage caused by fungi, slugs, nematodes and foliar feeding insects (Wang & Jeffers 2000;Jagdale & Grewal 2008;Zhen & Agudelo 2012). At the same time, there is increasing reluctance to apply potentially toxic agrichemicals to control pests and pathogens in the residential environment. ...
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A total of 84 bacterial endophytes were isolated from seeds of 6 cultivars of ornamental hostas, and they were identified to 5 species based on morphological characteristics and 16S rDNA sequence analysis. Among them, the strain ‘Blu-v2’, which was isolated from the seeds of cultivar ‘Blue Umbrella’ and identified to be Bacillus amyloliquefaciens, showed highest antifungal activity and capacity to deter feeding by Fall armyworms (Spodoptera fruigiperda). Lipopeptides in cultures of Blu-v2 were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its antifungal activities were verified. However, the lipopeptide preparation did not show toxicity to larvae of Fall armyworms. In a greenhouse experiment, Blu-v2 was inoculated into small plantlets of hosta (cultivar ‘Rainforest Sunrise’). The leaves of plants with bacteria (endophyte-infected = E+) and without bacteria (endophyte-free = E−) were used in seven-day feeding experiments employing fourth-instar larvae of Fall armyworms. We found that there was a significant decrease in the weights of larvae fed with E+ compared to E− plants; and the mortality rate of larvae fed with E− leaves was lower (3.33%) compared to that of larvae fed with E+ leaves (30%). Based on our studies, we suggest that endophytic B. amyloliquefaciens strain Blu-v2 has potential value as a biocontrol agent to reduce damage from fungal diseases and insect pests of hosta cultivars.
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Aphelenchoides pseudogoodeyi has recently been reported in association with seeds of forage grasses and rice in Brazil and senescent strawberry plants, in the United States. This nematode is likely a mycophagous species; however, so far, its pathogenicity potential to plants is unclear. This study aimed to verify the pathogenicity of A. pseudogoodeyi to two species of ornamental plants. The experiments were conducted by inoculating A. pseudogoodeyi onto Bird's-Nest Fern (Asplenium nidus) and Oriental Lily (Lilium speciosum) leaves, using two inoculation methods (with and without injury). After 40 days of inoculation (DAI) in Bird's-Nest Fern and 5, 10, 20 and 40 DAI in Oriental Lily, the pathogenicity and the host efficiency were evaluated by symptoms observation and by severity, final nematode population and reproductive factor (RF), respectively. Additionally, a histopathological study was performed by inoculating A. pseudogoodeyi onto Bird's-Nest Fern for observing anatomical alterations. A. pseudogoodeyi was able to cause local necrotic lesions on both Bird's-Nest Fern and Oriental Lily leaves. However, the presence of injury was essential to enable A. pseudogoodeyi to penetrate and cause those symptoms in both plant species. Also, the total population of A. pseudogoodeyi decreased drastically over time and RF was <1, which characterized these species as poor-host or resistant plants. A. pseudogoodeyi penetrated into the foliar tissue and induced a total destruction of the mesophyll and collapse of the cells, with the formation of large intercellular spaces. It is concluded that A. pseudogoodeyi is an opportunistic pathogen as injury was required to induce symptoms in Bird's-Nest Fern and Oriental Lily.
Several chemical, biopesticide and elicitor treatments were evaluated on healthy plants inoculated with leaf and bud nematodes (Aphelenchoides fragariae). Aphelenchoides fragariae is a major threat to ornamental plant production worldwide, and its management poses a great challenge due to its wide host range of herbaceous, ferns and woody plants. Commercially available products containing spirotetramat, abamectin, azadirachtin and the elicitor of plant defences, acibenzolar-S-methyl (ASM), were assessed for their ability to reduce the multiplication of A. fragariae inoculated on two plant species: Anemone hupenhensis (Japanese anemone), and Buddleja davidii (Buddleja). Treatments were applied individually and in combination with ASM in a spray programme. All treatments consistently showed a significant reduction of nematode multiplication in treated plants compared to the control. Programmes of ASM with spirotetramat, abamectin or azadirachtin had significantly lower nematode populations on both plant species compared to the untreated plants. In Buddleja plants, a programme using spirotetramat + ASM had a 97% reduction of the A. fragariae population over the control (ROC) while ASM, spirotetramat, abamectin, azadirachtin, ASM + abamectin and ASM + azadirachtin caused between 78 and 94% ROC. On Japanese anemone, the highest nematode ROC (95%) was obtained with the spirotetramat + ASM programme, while other treatments (ASM, spirotetramat, abamectin, azadirachtin, ASM + abamectin and ASM + azadirachtin) had a range of 80–94% reduction of A. fragariae over the control. Management of plants infested by leaf and bud nematodes may prove challenging; however, all the treatments investigated demonstrated a significant reduction in nematode population in Buddleja and Japanese anemone, indicating that they have significant potential as effective alternatives to manage A. fragariae in ornamental plants. The potential of ASM alone or in programmes with other actives is discussed in relation to their use in the management of A. fragariae.
This book describes methods for evaluating the resistance and tolerance of plant cultivars to important parasitic nematode species, such as root-knot, cyst and reniform, and discusses the concepts and consequences of resistance. This book provides an invaluable source of information to all plant pathologists, nematologists and plant breeders.
This book describes methods for evaluating the resistance and tolerance of plant cultivars to important parasitic nematode species, such as root-knot, cyst and reniform, and discusses the concepts and consequences of resistance. This book provides an invaluable source of information to all plant pathologists, nematologists and plant breeders.
Life cycle studies of Ditylenchus angustus demonstrated that one generation time from egg to egg took 8 days on one-week-old rice seedling at 24-26°C. The eggs were laid at 2-celled stage and embryos hatched in sterile water 64-66 hr after the egglaying. The duration of J2, J3 and J4 was 1, 1 and 2 days, respectively. Females started the egg-laying 1 day after the adulthood. Fecundity of D. angustus on rice plant was always higher than on the fungus, Botrytis cinerea. Fecundity declined on both hosts following the pre-culture on the fungi for 1 month (1MF) or for 6 months (6MF) prior to inoculation. D. angustus increased 1067, 993 and 734-fold on rice plant 40 days after inoculation with 20 adults (10 females and 10 males) collected from rice plant, 1MF and 6MF, respectively. On B. cinerea after the same period from the same initial population the multiplications were 291 and 229-fold, from the inoculum from rice plant and 6MF, respectively. Jpn. J. Nematol. 26 (1/2) 12-22 (1996).
The reproductive potential of the nematode Ditylenchus destructor was evaluated on two reported hosts, alfalfa (Medicago sativa) and red clover (Trifolium pratense), and three crops of unknown host status, corn (Zea mays), oat (Avena sativa), and snapbean (Phaseolus vulgaris), in microplot, greenhouse, and laboratory experiments. Potato and fallow treatments were included for comparison. Relative to the potato cultivar Norland, snapbean was a good host, red clover and corn were intermediate hosts, alfalfa was a poor host, and oat was a nonhost for nematodes grown in vitro in monoxenic cultures (.)
A direct infestation method as a means of rearing pure populations of A phelenchoides ritzemabosi is described. A mature female is placed directly on the ventral surface of the leaf on a tiny droplet or a thin film of water. Reproduction takes place after successful entry of the nematode into the leaf tissue. The progeny of this female is then transferred to new leaves and the procedure is continued. Methods of maintaining high humidity around the leaves are described. The use of 'mist propagation frames' to enhance the spread of disease in greenhouse beds is recommended.
The reproductive fitness of three Pratylenchus coffeae populations (Honduras, Ghana and Vietnam) and three Radopholus similis populations (Costa Rica, Cuba and Ghana) on carrot disks was studied as a function of time and inoculum densities. In the first study, the reproductive fitness of the isolates was followed during 11 weeks for P. coffeae and during 7 weeks for R. similis. All the populations increased with time. No distinction could be found in the maximum growth rate of the Pratylenchus populations. The R. similis population from Cuba had a higher maximum growth rate than those from Ghana and Costa Rica. All the Radopholus populations showed a faster multiplication than P. coffeae. In the second study, the influence of the inoculum density on the reproductive fitness was determined for the six populations. The differences in reproduction ratios confirmed the results of the first study.
This book summarizes the advances in nematology that have been made during the 20th century and provides perspectives for the development of nematology in the next century. Chapters comprise: plant diseases caused by nematodes; virus vectors; physiological interactions between nematodes and their host plants; taxonomy of insect parasitic nematodes; resistance to plant parasitic nematodes; crop rotation and other cultural practices as control strategies; use of antagonistic plants and natural products; biological control of nematodes by fungal antagonists; biological control of nematodes with bacterial antagonists; biological control of insects and other invertebrates; cost-benefits of nematode management through regulatory programmes; past and current uses of nematicides; and irradiation effects of plant parasitic nematodes.
Adults of Aphelenchoides ritzema-bosi tend to migrate up the stems of chrysanthemum plants in stationary water films possibly by a negatively geotropic response. A current of water down the stem opposes such an upward movement. Greatest mobility occurred in thick films of water in places with a high concentration of epidermal hairs as at the top of the stem and on the undersurface of leaves. Ciné films of movement in thick and thin films showed that there were fundamental differences in the type of locomotion in these two environments. Invasion of leaves via stomata was observed and the method of movement is described. The presence of A. ritzema-bosi in leaves appears to render the epidermis permeable to water. During dry weather there is little movement inside the leaf, but after rainfall activity increases as water enters the leaf. Spread of eelworm infestation in the leaf occurs in the mesophyll and across veins although initially these act as barriers. Emergence occurs via the stomata, chiefly on the undersurface. When the leaf is wet, about 50% of the eelworms emerge in the first hour. During wet weather many eelworms were recovered from the surfaces of leaves and it is suggested that eelworms spread mostly under these conditions.