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: firstname.lastname@example.org
Accepted for publication 6 May 2012.
http://dx.doi.org/10.1094 / 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
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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