Pre-inoculation with arbuscular mycorrhizal fungi suppresses root knot
nematode (Meloidogyne incognita) on cucumber (Cucumis sativus)
Zhang, L. D., Zhang, J. L., Christie, P., & Li, X. L. (2008). Pre-inoculation with arbuscular mycorrhizal fungi
suppresses root knot nematode (Meloidogyne incognita) on cucumber (Cucumis sativus).
Biology and Fertility of
Soils
,
45
(2), 205-211. https://doi.org/10.1007/s00374-008-0329-8
Published in:
Biology and Fertility of Soils
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SHORT COMMUNICATION
Pre-inoculation with arbuscular mycorrhizal fungi
suppresses root knot nematode (Meloidogyne incognita)
on cucumber (Cucumis sativus)
Lidan Zhang & Junling Zhang & Peter Christie &
Xiaolin Li
Received: 23 June 2008 / Revised: 19 August 2008 / Accepted: 20 August 2008 / Published online: 3 September 2008
#
Springer-Verlag 2008
Abstract A pot experiment was conducted to evalua te the
influence of pre-inoculation of cucumber plants with each
of the three arbuscular mycorrhizal (AM) fungi Glomus
intraradices, Glomus mosseae, and Glomus versiforme on
reproduction of the root knot nematode Meloidogyne
incognita. All three AM fungi tested significantly reduced
the root galling index, which is the percentage of total roots
forming galls. Numbers of galls per root system were
significantly reduced only in the G. in traradic es+M.
incognita treatment. The number of eggs per root system
was significantly decreased by AM fungus inoculation, no
significant difference among the three AM fungal isolates.
AM inoculation substantially decreased the number of
females, the number of eggs g
−1
root and of the number
of eggs per egg mass. The number of egg masses g
−1
root
was greatly reduced by inoculation with G. mosseae or G.
versiforme. By considering plant growth, nutrient uptake,
and the suppression of M. incognita together, G. mosseae
and G. versiforme wer e more effective than G. intraradices.
Keywords Glomus spp.
.
Nematode reproduction
.
Cucumber
Introduction
Cucumber is one of the economically most important
vegetables in China, the cultivated area of which (9.85×
10
5
ha) accounts for 5.4% of the total vegetable cultivation
area (Chines e Ministry of Agr iculture 2006). In recent years
yield losses have been strongly associated with root knot
nematode (RKN) caused by Meloidogyne spp. together
with soil pathogens such as Fusari um oxysporum, Phytoph-
thora melonis, and Pseudoperonospora cubensis (Qu et al.
2003). These soil pathogens are wides pread and may
become the major constraint in many cucumber production
areas, particularly in greenhouse systems in which contin-
uous cropping is practiced. A 3-year survey conducted in
Shandong province, northeast China, found RKN disease in
67.6% of greenhouses investigated and approximately 50%
of the plants infected (Dong et al. 2004). After four or more
crops, the rate of occurrence of RKN was up to 95% (Dong
et al. 2004) and RKN has been associated with fruit yield
losses of 20–30% and even 100% in some cases (Peng
1998). Unlike F. oxysporum, P. melonis and P. cubensis
which can be controlled to some extent by means of
grafting or the use of resistant cultivars, there is still no
satisfactory control measure for RKN disease in continuous
cropping of cucumber in China. Chemical nematicides are
prohibited due to their potentially detrimental effects on the
environment and human health even though they have been
Biol Fertil Soils (2008) 45:205–211
DOI 10.1007/s00374-008-0329-8
L. Zhang
:
J. Zhang (*)
:
P. Christie
:
X. Li
Key Laboratory of Plant Nutrition, Ministry of Agriculture,
China Agricultural University,
Beijing 100094, China
e-mail: junlingz@cau.edu.cn
L. Zhang
:
J. Zhang
:
P. Christie
:
X. Li
Key Laboratory of Plant–Soil Interactions, Ministry of Education,
China Agricultural University,
Beijing 100094, China
L. Zhang
:
J. Zhang
:
P. Christie
:
X. Li
Department of Plant Nutrition,
College of Agricultural Resources and Environmental Sciences,
China Agricultural University,
Beijing 100094, China
P. Christie
Agricultural and Environmental Science Department,
Queen’s University Belfast,
Newforge Lane,
Belfast BT9 5PX, UK
shown to be effective in inhibiting nematode infestations.
Agronomic practices such as crop rotations and the use of
fallow and cover crops could be employed but have not
been introduced in China because of economic factor.
Biological control of nematodes is therefore an attractive
option with the aim of maintaining current cultivation
practices and minimizing damage to the envir onment.
Arbuscular mycorrhizal (AM) fungi are ubiquitous soil
organisms that can form mutualistic associations with the roots
of the majority of vascular plant species (Lindermann 1988).
The establishment of AM association is often beneficial for
plant nutrition and AM fungi may also confer tolerance of
and resistance to various abiotic and biotic stresses to the host
plant(SmithandRead1997; Colla et al. 2008). It has even
been suggested that many root pathogens can be regarded as
pathogens of mycorrhizae (Rhodes 1980), implying that AM
fungi are closely associated with soil-borne pathogens,
including nematodes (Graham 2001). Since AM fungi and
RKN are all indigenous soil organisms and therefore co-exist
in plant roots, the potential role of AM fungi as biocontrol
agents and their protective effects on plants against RKN
have been well documented (Hussey and Roncadori 1982;
Diedhiou et al. 2003). Studies have shown that inoculation
with AM fungi can significantly reduce RKN infestation and
reproduction in some plant–nematode systems (Hol and
Cook 2005) such as papaya with Meloidogyne incognita
(Jaizme-Vega et al. 2006), olive planting stocks with M.
incognita and Meloidogyne javanica (Castillo et al. 2006),
tomato with M. incognita (Talavera et al. 2001; Siddiqui and
Akhtar 2007), pyrethrum with Meloidogyne hapla (Waceke
et al. 2001), Prunus rootstocks with M. javanica (Calvet et al.
2001), and banana with M. javanica (Rodriguez and Jaizme-
Vega 2005). In addition to enhancement of plant nutrition,
especially P nutrition, establishment of arbuscular mycorrhi-
zae may exert beneficial effects on plant growth though direct
competition with RKN for infection sites and space, alteration
of the composition of root exudates, or through activation of
plant defense reactions and other mechanisms (Smith et al.
1986; Azcón-Aguilar and Barea 1996; Harrier and Watson
2004;Lietal.2006). However, the positive response in
interactions between AM fungi and nematodes can vary and
negative responses and lack of any response have also been
reported (Smith et al. 1986; Carling et al. 1989).
Inoculation of cucumber plants with AM fungi has been
shown to increase plant growth (Trimble and Knowles
1995), protect p lants from salt stress (Ro sendahl and
Rosendahl 1991), and improve plant resistance to Fusarium
wilt (Hao et al. 2005) and Pythium ultimum (Rosendahl and
Rosendahl
1990). It may therefore be feasible to use AM
fungi in the control of RKN in cucumber production. The
present study was conducted to assess the possible effects
of three Glomus species on cucumber growth and nematode
infection and reproduction.
Materials and methods
Host plants and soil
Cucumber seeds (Cucumis sativus L. cv. Zhongnong 16)
were surface sterilized with a 10% (v/v)solutionofH
2
O
2
for
10 min and then rinsed thoroughly with deionized water. The
seeds were placed on autoclaved filter papers soaked with
sterile distilled water and incubated at 25°C for 24 h. Four
seedlings were planted in each pot and were thinned to two
per pot after the emergence of the second leaf.
Soil was collected from Daxing county in the suburbs of
Beijing and was a low phosphorus loamy sand with the
following physico-chemical properties: total N 0.08%, avail-
able P (Olsen-P) 7.72 mg kg
−1
, available K (NH
4
OAc-K)
33.6 mg kg
−1
,pH(H
2
O, soil 2.5:1, v/v)8.44,andtotal
organic matter 0.30%. Soil and river sand were sieved
(<2 mm) and sterilized by autoclaving at 121°C for 2 h.
After drying at room temperature, the soil and river sand
were mixed at a ratio of 1:1 (v/v). Before use the substrate
was amended with a mixture of nutrients as basic fertilizers:
N 200 mg kg
−1
(NH
4
NO
3
), P 50 mg kg
−1
(KH
2
PO
4
),
K 200 mg kg
−1
(K
2
SO
4
), Mg 100 mg kg
−1
(MgSO
4
7H
2
O),
Zn 5 mg kg
−1
(ZnSO
4
.
7H
2
O), Mn 5 mg kg
−1
(MnSO
4
7H
2
O)
and Cu 5 mg kg
−1
(CuSO
4
.
5H
2
O).
Arbuscular mycorrhizal fungal isolates
Three AM fungal species, Glomus intraradices Schenck &
Smith (BEG141), Glomus mosseae (Nicol & Gerd) Gerd &
Trappe (BEG167), and Glomus versiforme (Karsten) Berch
were propagated on maize and white clover host plants in a
growth chamber at 30°C/18°C with a 16 h/8 h light/dark
regime and 50–75% relative humi dity. The plants were
harvested after growth for 4 months. Soils containing
spores, external mycelium and roots of maize and white
clover were used as inocula.
The experimental containers were plastic pots 16 cm in
diameter×14 cm in height. Soil and sand mixture (1.5 kg)
and 150 g inoculum were placed in each mycorrhizal pot.
The inoculum was placed about 2 cm under the soil surface.
Non-mycorrhizal pots received an equivalent amount of soil
and sterilized inoculum together with 10 ml soil filtrate
(0.45 μm pore size) to provide a similar soil microbial
community (Calvet et al. 1993).
Nematode isolates
M. incognita was kindly provided by Professor Z. P. Cao of
the College of Resources and Environmental Sciences,
China Agricultural University, Beijing . The nemato des
were multiplied on tomato (Lycopersicon esculentum cv.
‘Hezuo 908’) using single egg mass inoculation. Plants
206 Biol Fertil Soils (2008) 45:205–211
were placed in a greenhouse at the Department of Plant
Nutrition, China Agriculture University and grown for
3 months. Severely infected roots were harvested and
washed with deionized water. The egg masses were hand-
picked using sterilized tweezers and the nematodes were
then collected according to the method of Hussey and
Barker (1973). Briefly, the detached eggs from the roots
were extracted in 1.5% NaClO. The eggs were added to
deionized water by using a 25-μm sieve and stirred to
stimulate the development of J2 juveniles. The separation
of eggs and juvenile nematodes then followed the method
of Baerman (Oostenbrink 1960). The concentration of the
suspension of nematodes was adjusted to 400 J ml
−1
. After
growth for 30 days, cucum ber plants wer e inoculated with
2,000 (J2) nematodes per pot by injecting the suspension
into five holes of 3-cm depth. The holes were formed in a
circle about 2 cm away from the stem base of the seedlings.
Experimental treatments and plant growth conditions
There were eight treatments: (1) non-inoculated control (-AMF-
Mi); (2) non-inoculated control, inoculated 30 days later with
M. incognita (−AMF+Mi); (3) inoculated with G. intraradices
(+Gi); (4) inoculated with G. intraradices, inoculated 30 days
later with M. incognita (+Gi+Mi); (5) inoculated with G.
mosseae (+Gm); (6) inoculated with G. mosseae, inoculated
30 days later with M. incognita (+Gm+Mi); (7) inoculated
with G. versiforme (+Gv); (8) inoculated with G. versiforme,
inoculated 30 days later with M. incognita (+Gv+Mi).
Each treatment was replicated eight times. M. incognita
inoculum was applied 30 days after planting when the
fourth leaf had emerged. Seedlings were grown for a further
5 weeks and then harvested. The pots were arranged in a
completely random ized design in a greenhouse at China
Agriculture University in Beijing. Seedlings were grown
from April to June at a temperature regime of approxi-
mately 35°C/23°C (day/night) with a 16 h/8 h (light/dark)
photoperiod and 50–75% relative humidity. Seedlings were
irrigated daily with deionized water and weekly with P-free
Hoagland nutrient solution (Liu and Li 2000)after
inoculation with M. incognita.
Harvest and analysis
At harvest shoot length, leaf numbers, shoot dry weight,
root fresh weight, number of galls, reproduction of
nematodes, mycorrhizal infection, and shoot concentrations
of nutrient elements were determined. Fresh roots were
divided into three pa rts: a portion was used to record the
egg masses and eggs, a portion to count the females, and
the remainder to measure root length an d the percentage of
root length colonized by AM fungus. The severity of RKN
disease was denoted by root galling index which was
assessed on a rating scale of 0–4 according to the number
of roots forming galls expressed as a proportion of the total
root system: 0=no galls,1 =1–25%, 2=26–50%, 3=51–
75%, and 4=76–100% (Krusberg and Nelson 1958). The
number of galls on the roots was recorded using an
arithmometer. Egg masses were stai ned in 0.015% phloxine
B for 20 min, rinsed in sterilized distilled water and then
counted under a stereomicrosco pe. Eggs were estimated
according to Hussey and Barker (1973). The egg suspen-
sionwasadjustedto25mland1mlofthesuspension
wastakenandusedtorecordthenumberofeggs.The
number of female nematodes within the roots was counted
using a dissecting microscope (magnification, ×40) and
staining by the NaOCl-acid fuchsin technique (Byrd et al.
1983). The percentage of root length colonized by AM fungi
was determined using the grid line inte rsect meth od
(Giovannetti and Mosse 1980) under a stereoscopic micro-
scope. Plant shoots were dried in an air-forced oven at 70°C
for 48 h. The dried samples were milled with a high-speed
micro-pulverizer (Whirl Type, ModelY-60, Hebei, China)
prior to elemental analysis. Sub-samples (about 0.3 g) were
wet digested with HNO
3
and H
2
O
2
in a microwave digester
(MARS CXPress, CEM Corporation) and the concentrations
of the elements were measured by ICP-MS (Optima
3300DV, Perkin Elmer).
Statistical analysis
Analysis of variance was carried out using the SAS
software package version 6.12 (SAS Institute, Inc., Cary,
NC, USA). Duncan's multiple range test or Fisher's LSC
test was used to test for significant differences between
treatment means at the 5% level.
Results and discussion
No AM fungus colonization was observed on the roots of
uninoculated plants. Roots of inoculated plants were exten-
sively mycorrhizal and the mean percentage of root length
colonized ranged from 52% to 68% (Table 1). Root
colonization rates in the absence of M. incognita were
significantly higher in plants inoculated with G. mosseae or
G. intraradices than in those inoculated with G. versiforme
and in the presence of M. incognita were higher in plants
inoculated with G. intraradices than in those inoculated with
either of the other two fungi. Inoculation with M. incognita
decreased root colonization rates significantly in plants
inoculated with G. mosseae (by 16%) or G. versiforme (by
11%). Changes in AM fungus colonization in the presence of
nematodes has been observed in other studies (Carling et al.
1989; Waceke et al. 2001; Castillo et al. 2006) and has been
attributed mainly to competition between AM fungi and
Biol Fertil Soils (2008) 45:205–211 207
RKN for feeding sites and carbon substrates from host
photosynthesis (Smith 1998; Hol and Cook 2005).
Inoculation with M. incognita significantly increased
shoot length and root fresh weight but decreased root length
(Table 1) and shoot dry weight remained unaffected. Galls
in the roots might have caused the increase of root fresh
weight in M. incognita infested plants. Inoculation with the
RKN had significant effects on shoot Mg, Cu, and Zn
concentrations (Table 2) and frequently incre ased shoot Mg
and Cu concentrations but decreased Zn concentrations
irrespective of the AM fungal isolate present. Root knot
nematode infection often reduces plant growth and yield
Table 2 Elemental concentrations in shoots of mycorrhizal and non-mycorrhizal cucumber with or without M. incognita
Inoculation
treatment
P (mg g
−1
) K (mg g
−1
) Ca (mg g
−1
) Mg (mg g
−1
) Fe (mg kg
−1
) Mn (mg kg
−1
) Cu (mg kg
−1
) Zn (mg kg
−1
)
Without M. incognita
Non-mycorrhizal 4.86±0.19b 33.50±0.91b 29.43±0.79ab 11.16±0.35b 150.23±6.18b 26.99±1.11b 21.54 ±0.64b 27.34±1.37a
G. intraradices 4.17±0.13c 35.99±1.42ab 30.57±0.66a 12.82±0.19a 172.36±7.46a 28.46±0.98ab 17.94±0.16c 28.63±1.16ab
G. mosseae 5.25±0.17b 35.46±0.78ab 29.10±0.60ab 12.86±0.24a 158.13±5.87ab 29.44±0.74ab 24.45±0.45a 28.23±0.59ab
G. versiforme 5.77±0.15a 38.65 ±1.33a 27.85±0.90b 12.07±0.45ab 152.11±6.26b 30.89±1.61a 25.80±0.65a 31.47±1.35a
With M. incognita
Non-mycorrhizal 4.46±0.19c 31.61±1.54b 29.45±0.57a 12.34±0.17b 146.10±6.69b 28.69±0.92 bc 23.20±0.40d 23.15±1.20c
G. intraradices 4.31±0.06c 36.34±1.76b 28.13±0.60a 12.75±0.25ab 156.91 ±6.58b 24.99±1.30c 18.00±0.17c 27.33±1.39ab
G. mosseae 5.40±0.17b 34.64±1.34b 28.56±0.71a 13.10±0.23ab 160.75 ±9.59b 32.37±1.18ab 25.29±0.44b 24.89±0.96bc
G. versiforme 6.25±0.14a 42.19±0.86a 30.05±0.99a 13.37±0.35a 194.12±6.85a 32.94±0.95a 28.31±0.61a 30.12±1.01a
Significance
a
due to
Mi inoculation NS NS NS ** NS NS *** **
AMF inoculation *** *** NS *** * *** *** ***
Interaction NS NS NS NS ** NS NS NS
Plants were pre-inoculated with one of three Glomus species or remained non-mycorrhizal for 30 days and were then inoculated with M. incognita
for an additional 5 weeks. Data are the means of eight replicates±SE and were compared by Duncan's multiple range test. Within each group of
four values any two means sharing a lower case letter are not significantly different within a M. incognita treatment
NS Not significant
***P<0.001; **P<0.01; *P<0.05
a
By analysis of variance
Table 1 Shoot length, shoot dry weight, root fresh weight and root length of mycorrhizal and non-mycorrhizal cucumber plants inoculated with
or without Mi
Inoculation treatment Root colonization rate (%) Shoot length (cm) Shoot dry weight (g) Root fresh weight (g) Root length (m)
Without M. incognita
Non-mycorrhizal 0 27.5±3.6b 2.51±0.35b 5.56±0.53b 6.49±0.89c
G. intraradices 68±2a 24.3±1.7b 2.26±0.13b 5.32±0.24b 8.44±0.46c
G. mosseae 68±3a 51.9±3.7a 4.63±0.20a 12.1±0.63a 12.82±0.86b
G. versiforme 64±3b 53.2±3.2a 4.82±027a 14.08±0.94a 15.67±1.25a
With M. incognita
Non-mycorrhizal 0 31.0±3.0c 2.74±0.25c 9.16±0.48b 4.55±0.29c
G. intraradices 62±3a 34.4±4.8c 2.82±0.45c 10.16± 0.92ab 6.10±0.63c
G. mosseae 52±4b 57.3±2.4b 4.52±0.26b 14.04±0.64ab 9.74±0.65b
G. versiforme 53±4b 73.7±2.8a 5.66±0.16a 17.54± 0.79a 11.58±0.81a
Significance
a
due to
Mi inoculation *** *** NS *** ***
AMF inoculation *** *** *** *** ***
Interaction * NS NS NS NS
Data are the means of eight replicates±SE and were compared by Duncan's multiple range test. Within each group of four values any two means
sharing a lower case letter are not significantly different within a M. incognita treatment
NS Not significant
***P<0.001; **P<0.01; *P<0.05
a
By analysis of variance
208 Biol Fertil Soils (2008) 45:205–211
