Available via license: CC BY-NC-ND 4.0
Content may be subject to copyright.
S. Afr. J. Enol. Vitic., Vol. 41, No. 1, 2020
*Corresponding author: E-mail address: knoetzer@arc.agric.za
Acknowledgements: The authors would like to thank the Agricultural Research Council, Winetech and the South African Table Grape Industry for funding the
research, and B. Sokomani, D. Hinds and C. Paulse for technical assistance
The Effect of Hot Water Treatment of Rooted Grapevine Nursery
Stock on the Survival of the Root-knot Nematode, Meloidogyne
javanica (Nematoda: Heteroderidae)
R Knoetze*
Agricultural Research Council, Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa
Submitted for publication: August 2019
Accepted for publication: November 2019
Key words: Grapevine, Meloidogyne, hot water treatment, root-knot nematode, control
Root-knot nematodes (Meloidogyne spp.) are endoparasites which cause severe losses in grapevine. To
ensure economically viable grape production, it is important that nurseries produce rooted nursery material
free of plant-parasitic nematodes. Hot water treatment (HWT) at 50°C for 45 min to eliminate root-knot
nematode (RKN) from rooted nursery material was investigated as a method to ensure nematode free
plant material. Rooted grapevine rootstocks known to be susceptible (US 8-7 and 110 Richter), moderately
resistant (1103 Paulsen and 143 B) and resistant (Ramsey) to Meloidogyne javanica were articially
infested by inoculating them with RKN eggs and larvae. After one growing season, the vines were lifted,
shoots and root systems trimmed and subjected to different HWT regimes viz. 50°C for 45 min and 55°C
for 20 min, while some were left as untreated controls. To evaluate plant response, each vine was planted
in a pot, together with a three-week old tomato seedling as an indicator of root-knot nematode infestation.
The tomato plants were removed after 12 weeks and their roots examined for the presence of M. javanica
galls and egg masses. At the end of the growing season, the effects of the treatments on plant growth were
assessed by determining total shoot and root mass. The results demonstrated that HWT at 55°C for 20 min
signicantly reduced the nematode populations in the rooted stocks, but did not eliminate the nematodes
from the roots since indicator plants from HWT vines still supported a small number of galls. HWT at
55°C for 20 min also reduced the level of infestation of RKN in grapevine planting material, but resulted
in a signicant reduction in growth.
INTRODUCTION
Root-knot nematodes (Meloidogyne spp.) (Tylenchida:
Heteroderidae) are endoparasites which cause severe
losses in grapevine. They penetrate the roots to feed,
inducing the formation of characteristic root galls. Heavy
infestation weakens the root system, restricting the ability
of roots to absorb water and nutrients, leading to reduced
vigour and yield (Storey et al., 2017). Nematode control in
established vineyards is costly and untreated infestations
can reduce the productive lifespan of a vineyard. Providing
nematode-free planting material to growers is key to ensure
economically viable grape production. Although nurseries
use soil fumigation and chemical control throughout the
year, endoparasites like root-knot nematodes (RKN) can still
be transmitted by infested rooted plant material.
Hot water treatment (HWT) is an effective and practical
method for the control of a number of grapevine pests
and diseases in dormant grapevine cuttings and young
rooted vines (Von Broembsen & Marais, 1978; Suatmadji,
1982; Loubser & Höppner, 1986; Fourie & Halleen, 2004;
Gramaje et al., 2009). Barbercheck (1986) showed that HWT
eliminated root-knot nematodes from grapevine nursery
stock, but concerns have been raised that this HWT regime
may not be sufcient to ensure that no viable RKN adults,
juveniles or eggs survive in the roots of treated plants.
The South African Plant Certication Scheme for wine
grapes requires plant material to be visually free of RKN
infestation for certication, whilst HWT for eradication
of RKN in rooted grapevine material is not prescribed in
the Standard Operating Procedure (SOP) for Grapevines
pertaining to rejections of graft combinations for visual
symptoms of RKN (https://www.gov.za/documents/plant-
improvement-act-south-african-plant-certication-scheme-
wine-grapes-amendment). The current SOP does prescribe
HWT at 50°C for 45 min to eliminate crown gall and aster
yellows phytoplasma, or HWT at 55°C for 5 min to remove
other supercial pathogens and pests. Conrmation of the
efcacy of HWT at 50°C for 45 min for the elimination of
RKN from rooted vines is important for the Vine Improvement
DOI: https://doi.org/10.21548/41-1-3705
S. Afr. J. Enol. Vitic., Vol. 41, No. 1, 2020
Hot Water Treatment of Grapevine for the Control of Root-knot nematodes
Association before revising current regulations and SOPs.
The aim of this study was to test the efcacy of HWT
for eliminating RKN from rooted nursery material, as well as
the impact of HWT on the initial growth of the treated plants.
MATERIALS AND METHODS
Inoculation of grapevine rootstocks
Grapevine rootstocks known to be susceptible (US 8-7 and
110 Richter), moderately resistant (1103 Paulsen and 143 B)
and resistant (Ramsey) to Meloidogyne javanica (Treub,
1885) Chitwood, 1949 were grafted with Chenin blanc,
callused and rooted at a commercial grapevine nursery
(Vititec, Paarl). Rooted vines were planted in sandy soil in
planting bags and kept in a glasshouse, set at 25°C. Twenty-
one representatives of each rootstock were inoculated
with M. javanica on four occasions during the 2017/18
growing season by pipetting 5 ml of a suspension containing
approximately 3000 eggs and juveniles into two holes made
in the soil, approximately 1.5 to 2.0 cm from the plant stem.
Inoculation sites were covered by pressing the soil mixture
back into place. Seven representatives of each rootstock
were not inoculated (control plants) and kept separately to
avoid contamination. After the rst growing season, root
samples were collected and pooled for each rootstock type,
where-after 3 x 10 g subsamples were used to determine the
level of infestation. The number of eggs per gram of roots
was determined by using Riekert’s adapted NaOCl method
(Riekert, 1995), whereby the roots were cut into 10-mm
pieces and shaken thoroughly for 4 min in a 1 % NaOCl
solution. The solution was decanted through stacked sieves
(75- and 25-μm-aperture) and washed thoroughly before the
eggs and juveniles were collected from the 25-μm-aperture
sieve.
Hot water treatment
After the rst growing season, the vines were lifted, shoots
trimmed to three buds and the root systems trimmed to
approximately 150 mm. The plants were then packed into
plastic bins, covered with sawdust and stored in a cold
room at 5°C. Plants from each rootstock were divided into
4 groups, constituting the different treatments, which were:
i) infected plants, no HWT; ii) infected plants, HWT at
50 °C for 45 min; iii) infected plants, HWT at 55 °C for
20 min and iv) non-infected plants; no HWT. There were
seven replications of each treatment. Plants were submersed
in thermostatically controlled hot water baths, set at the
predetermined temperatures. Immediately after treatment
the vines were immersed in a cold water bath at 15°C for
15 min. After treatment, the vines were planted immediately
and moved to a glasshouse at 25°C.
Evaluation of the efcacy of HWT
To evaluate treatment efcacy, each vine was planted in a
plastic pot containing sterilized sandy soil and placed in a
glasshouse at 25°C. The pots were arranged in a randomized
block design, consisting of seven blocks, with 20 pots in
each block. The vines were drip-irrigated with ltered water
(5 µm lter) to avoid external contamination, as well as cross
contamination between pots. Three days later, a three-week
old tomato seedling (cv. Moneymaker) was planted next to
each vine as indicator of RKN infestation.
Twelve weeks later, the tomato plants were removed,
their roots stained with a 0.1 % food dye containing Ponceau
4R (Damasceno et al., 2016) for easier detection and then
examined for the presence of root-knot nematode galls and
egg masses. The number of galls and/or egg masses resulting
from each grapevine-tomato combination was recorded.
A value of 100 galls was assigned to heavily infested root
systems, as it was not possible to accurately count the
number of galls present.
Evaluation of growth response
To evaluate plant response to the HWT, budding and
sprouting of the plants after planting were monitored on a
weekly basis. Five months after planting (February 2019) the
grapevines were removed from their pots, leaves removed
and the root systems rinsed with water to be free of soil. Shoot
length and fresh shoot weight were measured immediately,
but the root weight was determined after overnight (12 h)
drying at room temperature.
Data analyses
The experimental design was a randomised block design
with seven block replications. The treatment design was
a factorial with two factors, rootstocks with ve levels
and hot water treatment with four levels. To interpret the
number of galls and/or egg masses on tomato root systems
the data was subjected to an ANOVA using General Linear
Models Procedure (PROC GLM) of SAS software (Version
9.4; SAS Institute Inc, Cary, USA). The kurtosis value and
symmetrical histogram in the Univariate Procedure (PROC
UNIVARIATE) of SAS Software indicated normality of
standardized residuals. Fisher’s least signicant difference
was calculated at the 5% level to compare treatment means
(Ott & Longnecker, 2001). To analyse growth variables of
infested plants, the data was subjected to an ANOVA using
General Linear Models Procedure (PROC GLM) of SAS
software (Version 9.4; SAS Institute Inc, Cary, USA). The
Shapiro-Wilk test conrmed normality of the standardized
residuals (Shapiro & Wilk, 1965). Fisher’s least signicant
difference was calculated at the 5% level to compare
treatment means (Ott & Longnecker, 2001).
RESULTS AND DISCUSSION
Infection rates
The grapevines were successfully infested with M. javanica
during the rst growing season. Table 1 shows the number of
eggs gram roots-1 detected at the end of the growing season.
The results conrmed that US 8-7 and 110 Richter were
the most susceptible to M. javanica, with 1103 Paulsen and
143 B being moderately susceptible and Ramsey the least
susceptible.
Efcacy of HWT
Data analysed with a factorial ANOVA showed signicant
differences (p < 0.0001) when comparing the number of
galls and/or egg masses on roots of tomato indicator plants,
planted next to M. javanica infected grapevines subjected
to different hot water regimes (Table 2). The presence of
nematodes in a tomato plant was considered proof that the
DOI: https://doi.org/10.21548/41-1-3705
S. Afr. J. Enol. Vitic., Vol. 41, No. 1, 2020
Hot Water Treatment of Grapevine for the Control of Root-knot nematodes
HWT did not successfully eradicate all the RKN present in
the roots of the grapevine plant. The number of RKN that
survived in all of the HWT plants was signicantly lower
than in the untreated control, but it was not zero, except in
the vines grafted onto Ramsey rootstocks. This means that
a small number of the nematodes was not killed by the
treatments and were able to produce eggs which could hatch
and infest the tomato plants. Suatmadji (1982) also found
that control by HWT (51.7°C for 5 min) was incomplete, as
indicator plants planted next to HWT vines supported a small
number of galls. This was attributed to the fact that immature
nematodes were imbedded in young galls consisting of
tightly packed cells, which may contribute to the nematodes
surviving the treatment. Barbercheck (1986) found that
HWT eliminated M. javanica from grapevine nursery stock,
but it must be considered that, in that case, tomato plants
were not used as indicators of RKN viability.
Eradication of the nematodes in the Ramsey material is
attributed to the fact that the material was the least infested
before the commencement of HWT. Thus, the combination
of plant resistance and HWT seems to have contributed to
effective eradication of the nematodes on this occasion.
Conversely, the combination of the most susceptible rootstock
(US 8-7) with the treatment of 50°C for 45 min, was the least
effective of the treatment combinations (Table 2).
Considering the combined treatment averages of all the
rootstocks, it is evident that the treatment of 55°C for 20 min
was signicantly more effective in reducing the nematode
population in infested rootstocks than the 50°C for 45 min
treatment (Table 2).
Growth response of vines to HWT
Cumulative sprouting percentages of the vines over a 7-week
period indicated that untreated plants started sprouting in
the rst week after planting, whilst treated plants started
sprouting in the second week. Overall, budburst of plants
treated at 50°C for 45 min was delayed by one week, whilst
budburst of plants treated at 55°C for 20 min was effectively
delayed for two weeks. The lowest sprouting percentage was
recorded for vines grafted onto Ramsey rootstocks treated
at 55°C for 20 min (57%), while all the vines grafted onto
Ramsey rootstocks treated at 50°C for 45 min sprouted
within 3 weeks.
Data analysed with a factorial ANOVA showed no
signicant differences between the growth responses of
different rootstocks, but the differences between treatments
were highly signicant (p < 0.0001). Table 3 shows the
plant responses with regard to shoot length, shoot weight
and root weight, regardless of rootstock. The best growth
was observed in plants treated at 50°C for 45 min, but it did
not differ signicantly from infected plants that were not
subjected to HWT. Studies carried out by Graham (2007)
showed that cuttings grown in cool climates in New Zealand
were susceptible to damage at 50°C for 30 min, but evidence
suggests that tolerance of plants to HWT is affected by the
climate in which the cuttings are grown (Waite & Morton,
2007). Von Broembsen & Marais (1978) found that treatment
for 15 min to 60 min at 50°C resulted in no phytotoxic effects
to dormant vines, but Loubser & Höppner (1986) reported
that plant mass increase as well as root mass of HWT (50°C
for 15 min) vines were signicantly lower than those of
untreated plants.
Shoot lengths and shoot weights of infested plants
treated at 50°C for 45 minutes were signicantly
more than those of uninfested, untreated plants. This
anomalous result can be attributed to the fact that closer
TABLE 1
Infestation rates of different rootstock types with Meloido-
gyne javanica.
Rootstock
Average RKN eggs
gram roots-1
US 8-7 145.0
Richter 110 49.3
1103 Paulsen 10.7
143-B 9.0
Ramsey 8.7
TABLE 2
Number of galls/egg sacs detected on roots of tomato indicator plants, planted next to Meloidogyne javanica infected grapevines,
subjected to different hot water regimes. Data are means of seven replications. Means in the same column followed by the same
letter do not differ signicantly according to Fisher’s LSD test (P > 0.05).
Treatment US 8-7 110 Richter 1103 Paulsen 143 B Ramsey
Combined
treatment
average
Infested
No HWT
*100.00 a *100.00 a *100.00 a *100.00 a 16.14 bc 83.23 a
Infested
50°C for 45 min
29.00 b 0.86 d 0.86 d 8.14 cd 0.00 d 7.77 b
Infested
55° for 20 min
1.43 d 0.00 d 0.29 d 0.14 d 0.00 d 0.37 c
Not infested
No HWT
0.00 d 0.00 d 0.00 d 0.00 d 0.00 d 0.00 c
*A value of 100 was assigned to heavily infested root systems, as it was not possible to accurately count the number of galls present.
DOI: https://doi.org/10.21548/41-1-3705
S. Afr. J. Enol. Vitic., Vol. 41, No. 1, 2020
Hot Water Treatment of Grapevine for the Control of Root-knot nematodes
inspection of the vines revealed dark-brown to black
discoloration in the xylem of the root crown and basal
rootstock of the vines. Fungal isolation (performed at
ARC Infruitec-Nietvoorbij) detected the presence of the
pathogens Phaeoacremonium aleophilum Gams, Crous,
Wingeld & Mugnai (Diaporthales: Togniniaceae) and
Pleurostomophora richardsiae (Nannf.) Mostert, Gams &
Crous (Calosphaeriales: Pleurostomataceae), both of which
are associated with Petri disease of grapevines. Fourie &
Halleen (2004) found that HWT (30 min at 50°C) of dormant
nursery material were effective in reducing fungal infection
levels in nursery plants. It is therefore possible that HWT
reduced the severity of the fungal infection in these vines,
leading to the masking of any growth retardation that may
have resulted from HWT at 50°C for 45 min.
The combined growth of plants treated at 55°C for 20 min
was signicantly less than with all of the other treatments.
Gramaje et al. (2009), when investigating HWT against Petri
disease pathogens, found that there was little variability in
the percentages of sprouting and shoot weight after HWT,
with the exception of the HWT at 54°C in which the highest
reduction was obtained. In this study, it was evident from
sprouting percentages, shoot length, shoot weight and root
weight that vines were damaged by HWT at 55°C for 20 min.
CONCLUSIONS
This research showed that HWT at 50°C for 45 minutes
greatly reduced the level of infestation of RKN in grapevine
planting material, particularly when RKN infestation levels
were low, but it did not eradicate RKN in all instances. HWT
at 55°C for 20 min also reduced the level of infestation
of RKN in grapevine planting material, but resulted in a
signicant reduction in growth and therefore cannot be
recommend for the treatment of rooted grapevine nursery
material.
Since HWT at 50°C for 45 min did not completely
eliminate RKN from rooted material, the unqualied revision
of current regulations and operating procedures cannot
be recommended to the Vine Improvement Association.
However, HWT can be successfully implemented in nurseries
as an added measure to reduce nematode infestation, but only
if due consideration is given to the prevention of infestation
of rooted material with RKN. An integrated strategy for
TABLE 3
Mean shoot length, shoot weight and root weight of Meloidogyne javanica infested grapevines, subjected to different hot water
regimes. Means in the same column followed by the same letter do not differ signicantly according to Fisher’s LSD test
(P > 0.05).
Treatment Shoot length (mm) Shoot weight (g) Root weight (g)
Infested
50°C for 45 min
1222.54 a 11.79 a 91.17 a
Infested
No HWT
1111.73 ab 10.25 ab 88.89 a
Not infested
No HWT
1004.24 b 9.50 b 85.10 a
Infested
55° for 20 min
808.34 c 6.53 c 54.60 b
the proactive management of RKN in grapevine nurseries,
which includes practices such as the ltering of irrigation
water, sterilization of growing medium, general sanitation
practices and the use of HWT, is advocated to provide RKN-
free planting material, which will ultimately save costs for
nematode control in established vineyards and prolong the
productive lifespan of vineyards.
LITERATURE CITED
Barbercheck, M., 1986. Control of Meloidogyne javanica in dormant
grapevine nursery stock. Phytophylactica 18, 39-40.
Damasceno, J.C.A., Soares A.C.F., de Jesus F.N. & Castro J.M.C., 2016.
Root-knot nematode staining with articial food dyes. Nematoda 3, 1-5.
http://dx.doi.org/10.4322/nematoda.01816
Fourie, P.H. & Halleen, F., 2004. Proactive control of Petri disease of
grapevine through treatment of propagation material. Plant Dis. 88, 1241–5.
https://doi.org/10.1094/PDIS.2004.88.11.1241
Graham, A., 2007. Hot water treatment of grapevine rootstock cuttings
grown in a cool climate. Phytopathol. Mediterr. 46, 124.
Gramaje, D., Armengol, J., Salazar, D., López-Córtes I. & Garcıá-Jiménez
J., 2009. Effect of hot-water treatments above 50°C on grapevine viability
and survival of Petri disease pathogens. Crop Prot. 28, 280–285. https://doi.
org/10.1016/j.cropro.2008.11.002
Loubser, J.T. & Höppner, G.F., 1986. Control of lesion nematodes,
Pratylenchus spp., in grapevine nursery material by immersion in
fenamiphos solutions and hot water. South Afr. J. Enol. Vitic. 7, 3-5.
Ott, R.L. & Longnecker, M., 2001. An introduction to statistical methods
and data analysis 5th Edition. Belmont, California, Duxbury Press.
Riekert, H.F., 1995. An adapted method for extraction of root-knot nematode
eggs from maize root samples. Afr. Plant Prot. 1, 41–43.
Shapiro, S.S., Wilk, M.B. & Chen, H.J., 1968. A comparative study of
various tests for normality. J. Am. Stat. Assoc. 63(324), 1343-1372.
Storey, S.G., Malan, A.P. & Hugo, H.J., 2017. Nematode Pests of Grapevine.
In: Fourie, H., Spaull, V.W., Jones, R.K., Daneel, M.S. & de Waele, D.
(Eds.) Nematology in South Africa: A view from the 21st century. Springer
International, Switzerland, pp. 345 – 357. https://doi.org/10.1007/978-3-
319-44210-5
Suatmadji, R.W., 1982. Control of root knot nematodes, Meloidogyne
javanica, in rootstocks of grapevine, Vitis vinifera, by immersion in
nematicide solutions at different temperatures and in hot water. Nematol.
Mediterr. 10, 119–125.
DOI: https://doi.org/10.21548/41-1-3705
S. Afr. J. Enol. Vitic., Vol. 41, No. 1, 2020
Hot Water Treatment of Grapevine for the Control of Root-knot nematodes
Von Broembsen, S. & Marais, P.G., 1978. Eradication of Phytophthora
cinnamomi from grapevine by hot water treatment. Phytophylactica 10,
25–27.
Waite, H. & Morton, L., 2007. Hot water treatment, trunk diseases and other
critical factors in the production of high-quality grapevine planting material.
Phytopathol. Mediterr. 46, 5–17.
DOI: https://doi.org/10.21548/41-1-3705