Journal of Plant Pathology (2014), 96 (1), 183-188
Edizioni ETS Pisa, 2014
Karuri et al. 183
INTERACTION OF FUSARIUM OXYSPORUM f. sp. VASINFECTUM AND THE FUNGAL
FEEDING NEMATODE APHELENCHUS AVENAE ON BT COTTON
H.W. Karuri1, R. Amata2, N. Amugune3 and C. Waturu4
2 National
1 Embu University College, University of Nairobi, P.O Box 6-60100, Embu, Kenya
Agricultural Research Laboratories, Kenya Agricultural Research Institute, P.O Box 14733-00800, Nairobi, Kenya
3 School of Biological Sciences, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya
4 Horticultural Research Centre, Kenya Agricultural Research Institute, P.O. Box 220, Thika, Kenya
SUMMARY
The fungal feeding nematode Aphelenchus avenae
(APH) feeds on different species of fungi including Fusarium oxysporum f. sp. vasinfectum (FOV) that causes
wilt in cotton. The objective of this study was to assess
the interactions of FOV and APH on Bt cotton and its
isogenic counterpart (isoline) under greenhouse conditions. The treatments consisted of three levels, where Bt
cotton, isoline and HART 89M were inoculated with: (i)
APH alone; (ii) FOV alone and (iii) APH+FOV. Vascular discoloration, plant height, number of nodes, number
of bolls, fresh shoot and root weight were recorded 180
days after planting (dap). Foliar symptoms were recorded
throughout the growing season, and ELISA was used to
determine the presence of Bt protein in soil and roots at
180 dap. Whereas no Bt protein was detected in roots
and soil of HART 89M and isoline, it was found in Bt
cotton. The isoline was more susceptible to FOV and
APH + FOV than Bt cotton and HART 89M. FOV and
APH + FOV caused a reduction in plant height, number
of nodes, number of bolls, fresh shoot and root weight but
the decrease was greater in the FOV treatment. There was
also a higher reduction of growth parameters in the FOV
treatment than in APH. The number of nematodes in the
APH + FOV treatment of Bt cotton and isoline were not
significantly different. The isoline was more susceptible
than HART 89M to FOV.
Key words: Trophic interactions, Bt cotton, Aphelenchus, Fusarium oxysporum
INTRODUCTION
Interactions between plant parasitic nematodes and
pathogenic fungi result in serious yield losses compared
to single infections. Damage by Verticillium, Pythium and
Rhizoctonia is enhanced in cotton in the presence of Meloidogyne spp. Delay in seed emergence and an increase in
severity of nematode infection were reported in combined
Corresponding author: H.W. Karuri
E-mail: [email protected]
infections of Meloidogyne javanica and Rhizoctonia solani
in soybean. Severity of fusarium wilt in cotton is increased
in the presence of the reniform, sting, lance and lesion
nematodes (Davis et al., 2006).
Fusarium wilt of cotton is caused by Fusarium oxysporum f. sp. vasinfectum (FOV) the main symptoms being
wilting, stunting, chlorosis and die back. In the field, development of fusarium wilt is influenced by the virulence
of the fungus, susceptibility of the crop, soil type and
fertility, weather conditions and interactions with other
organisms, including nematodes. Fusarium wilt has been
reported in East Africa including Kenya where it reduces
yields and affects fibre quality (ICAC, 2003).
The nematode Aphelenchus avenae (APH) feeds on
more than 52 genera of fungi, many of which are plant
pathogens, making it important as a biological control
agent. APH consumes 6.1 ng of N from fungal biomass in
24 h, increases N mineralization and the release of carbon
dioxide (Ingham et al., 1985) thereby contributing to soil
fertility (Chen and Ferris, 1999). Synergistic and antagonistic associations between fungal-feeding nematodes and
fungi have been recorded (Ragozzino and D’Errico, 2011).
Although fungal feeding-nematodes parasitize fungi and
feed on their cytoplasm, thus decreasing fungal biomass
(Wolfarth et al., 2013), they can also coexist with fungal endophytes in a cultivation-based mutualism, where
the nematode favours the growth of its preferred fungus
(Baynes et al., 2012). In parasitic associations between
pathogenic fungi and fungivorous nematodes, disease severity is reduced (Ruess and Dighton, 1996).
Interactions between soil organisms are influenced by
the soil environment, especially at the interface between
roots and soil, due to the high microbial activity promoted
by root exudates (Freckman and Caswell, 1985). Transgenic insect-resistant crops influence microorganisms either directly through the Bt protein or indirectly through
pleiotropic effects. Bt proteins in the plants can move to
higher trophic levels through species that show moderate or total resistance to Cry proteins. Roots of Bt cotton
contain Cry proteins which affect fungal growth (Gupta
and Watson, 2004). Donegan et al. (1995) reported differences in the abundance of fungi between Bt and nonBt cotton and Shen et al. (2006) found differences in the
184
Interaction of F. oxysporum and A. avenae
fungal composition of Bt and non-Bt cotton at different
growth stages. Turrini et al. (2004) found that Bt maize
significantly reduces the hyphal growth of the arbuscular
mycorrhizal fungus Glomus mosseae. Moreover, fungi associated with Bt cotton were significantly different from
those of non-Bt cotton cultivars, as more species were observed in residues of Bt cotton than in those of non-Bt
cotton (Gupta and Watson, 2004)
Bt proteins in transgenic crops have different effects on
nematodes, which may affect their interactions with other
organisms (Hoss et al., 2004, 2008, 2011; Griffiths et al.,
2006). A decrease or increase of FOV or APH abundance
due to the direct or indirect effect of Cry proteins may
result in changes in disease severity of fusarium wilt in cotton and, in turn, this may influence the overall interaction
between FOV and APH. In addition, pleotropic effects
arising from insertion of the cry genes in Bt cotton may
affect the induction or repression of defence-related genes
when the plant is infected by FOV resulting in an increase
or decrease in the severity of wilt (Dowd et al., 2004). The
main objective of the present study was to assess the interaction of FOV-APH on Bt cotton and its isoline.
MATERIALS AND METHODS
Greenhouse experiments. Pots containing sterilized
soil (1:1 sandy: loam) were arranged in a completely randomized design in the greenhouse and they contained Bt
cotton (06Z604D), isoline (99M03) and HART 89M (local non Bt cotton cultivar). Bt cotton 06Z604D (Bollgard
II) seeds resulting from a re-transformation of Bollgard
I which contains the cry1Ac gene and Neomycin phosphotransferase type II (NPTII) selectable marker protein
were provided by Monsanto (USA). Bollgard II contains
also the cry2Ab2 gene and produces beta-D-glucuronidase
(GUS) marker protein (Monsanto, 2003). Comparisons
were made between Bt cotton and its isogenic counterpart
to test the effect of the Bt gene, while HART 89M was
compared with its isoline to test varietal effects. The treatments consisted of three levels, where Bt cotton, isoline
and HART 89M were inoculated with: (i) APH alone; (ii)
FOV alone and (iii) APH+FOV. The three treatment levels
were replicated four times with each replicate consisting
of 12 plants grown in separate pots. Uninoculated plants
served as controls. One seed was planted at the centre of
each pot after surface sterilization with 0.1% mercuric
chloride for 2 min. After emergence, 5,000 APH nematodes were inoculated in the rhizosphere of each plant.
Each plant in the FOV and APH+FOV treatment was also
inoculated with 10 ml of a FOV spore suspension containing 1 × 106 conidia per ml.
Vascular discoloration, plant height, number of nodes,
number of bolls, fresh shoot and root weight were recorded at 180 dap. Foliar symptoms were recorded throughout the growing season and were rated on a 0 to 5 scale
Journal of Plant Pathology (2014), 96 (1), 183-188
where: 0 = no foliar symptoms; 1 = chlorosis and/or wilt
restricted to cotyledons or first leaf; 2 = chlorosis and/or
wilt extending beyond the first leaf; 3 = moderate to severe
foliar symptoms usually with some abscised leaves; 4 = severe foliar symptoms on the entire plant; 5 = dead plant
(Ulloa et al., 2006). Vascular discoloration was measured
from cross and longitudinal sections of the stem at the soil
level and rated as follows: 1 = no discoloration; 2 = light
brown, streaky discoloration; 3 = continuous dark brown
discoloration; 4 = plant dead (Hao et al., 2009). At crop
maturity the population density of FOV and APH was also
determined. The presence of Bt protein in soil and roots
was determined using qualitative ELISA.
The number of colony forming units (CFU) per gram
of soil was determined by taking aliquots of 100 μl of the
suspension at 1:1, 1:10, and 1:100 dilutions and plating
them onto each of three plates of peptone-based pentachloronitrobenzene (PCNB) Fusarium selective medium
(Leslie and Summerell, 2006). After seven days incubation at 25±2°C, individual colonies were counted. To
determine the number of CFU per gram of fresh stem
tissue, a piece of stem, approximately 5 cm in length,
between the soil line and first leaf was weighed, surface-sterilized in 0.5% (v/v) sodium hypochlorite for
two min, and homogenized for two min in a blender
with 100 ml of distilled water. Aliquots of 100 μl of the
suspension at 1:1, 1:10, and 1:100 dilutions were plated
onto each of three plates of PCNB medium. After seven
days, individual colonies were counted. Nematodes were
extracted in all cases using a modified Baermann technique (Whitehead and Hemming, 1965). The greenhouse
trial was repeated once.
Preparation of inoculum. Spores from a week-old FOV
colonies were washed from the surface of potato dextrose
agar (PDA) plates. Dilutions were made with sterile distilled water, and final concentrations were determined
with a haemocytometre. Nematodes extracted from soil
were transferred to PDA plates on which Fusarium sp.
was growing for multiplication. Nematodes cultured on
the medium were extracted, collected on a 500-mesh sieve
to remove fungal spores, surface sterilized in 0.01% HgCl2
for two min, then washed three times with sterile distilled
water. Nematode cultures were renewed by pipetting 25
μl of a nematode suspension in sterile distilled water on
fungal colonies growing on PDA amended with antibiotics (1 ml/l of each penicillin, streptomycin, and tetracycline) to obtain monoxenic cultures without bacterial
contamination.
FOV pathogenicity test. To confirm the pathogenicity
of the FOV isolate used in the experiment, one-week-old
cotton seedlings were uprooted, rinsed with tap water,
trimmed to 2 to 3 cm in length, and immediately dipped
for 3 min into an aqueous suspension of FOV macro- and
microconidia. Inoculated seedlings were then planted in
Journal of Plant Pathology (2014), 96 (1), 183-188
Karuri et al. 185
Fig. 1. Mean values from greenhouse evaluations for foliar
symptoms caused by Fusarium oxysporum f. sp. vasinfectum
(FOV) and combined infections of FOV and Aphelenchus
avenae (APH + FOV) on Bt cotton, isoline and HART 89M.
(0 = no foliar symptoms, 1 = chlorosis and/or wilt restricted
to cotyledons or first leaf, 2 = chlorosis and/or wilt extending
beyond the first leaf, 3 = moderate to severe foliar symptoms
usually with some abscised leaves, 4 = severe foliar symptoms
on the entire plant, and 5 = dead plant).
Fig. 2. Mean values from greenhouse evaluations for vascular
discoloration caused by Fusarium oxysporum f. sp. vasinfectum
(FOV) and combined infections of FOV and Aphelenchus avenae (APH + FOV) on Bt cotton, isoline and HART 89M (1, no
discoloration; 2, light brown, streaky discoloration; 3, continuous dark brown discoloration; and 4, plant dead).
soil and placed in a greenhouse at 27 to 30°C. After three
weeks they were rated for foliar and vascular symptoms of
fusarium wilt.
exceed 0.15 and 0.35 respectively. The mean, blank-subtracted OD of the positive control wells was at least 0.2
and the coefficient of variance (CV) between the duplicate
positive control wells did not exceed 15%. The positive
control ratio was calculated by dividing the OD of each
sample extract by the mean OD of the positive control
wells. For Cry2Ab, if the positive control ratio calculated
for a sample was less than 1.0, the sample did not contain
Cry2Ab. In the Cry1Ac part of the test, if the positive control ratio was less than 0.5, the sample did not contain the
protein. Results are reported as absence or presence of Bt
protein.
Quantification of Bt protein in rhizosphere soil and
roots collected from the nematode-fungus interactions
trial. Rhizosphere soil and roots from Bt cotton, HART
89M and isoline treatment were collected at 180 dap during the first and second trial. A qualiplate ELISA kit for
Cry1A and Cry2A (AP 051) (EnviroLogix, USA) was used
to determine the presence or absence of Bt protein. One
gram each of soil and 0.5 g of root samples were used for
analysis of Cry1Ac and Cry2Ab2 protein by ELISA. Extraction buffer (1000 µl) was added to the samples which
were then ground and vortexed for 1 min. The mixture
was then shaken on an orbital shaker at 300 rpm for 60
min, vortexed for 2 min and centrifuged at 16,000 rpm
for 10 min. The supernatant (50 μl) was used for analysis
following the manufacturer’s instructions. The amount of
Cry2Ab2 and Cry1Ac was determined using a spectrophotometer (Benchmark, Bio-Rad, USA).
Statistical analysis. Treatment effects on different parameters were determined using ANOVA (GenStat 12.1).
Means were separated using Fischer’s least significant differences test. Differences at P < 0.05 level were considered statistically significant. Results from the two trials
were similar and the data were pooled for analysis. ELISA
results were interpreted according to the manufacturer’s
protocol where the mean optical density (OD) of the blank
wells in the Cry1Ac and Cry2Ab part of the test did not
RESULTS
Wilting, vascular discoloration, chlorosis and necrosis of the leaves were the main symptoms observed in
the treatments encompassing FOV inoculations. Bt and
isoline exhibited significantly different (F = 64.9 [1, 9];
P < 0.001) severity in foliar symptoms and a significant
treatment×treatment level interaction for vascular discoloration (F = 73.7 [1, 9]; P < 0.001). There was a significant
treatment×treatment level interaction for foliar symptoms (F = 205.5 [1, 9]; P < 0.001) and vascular discoloration
(F = 460.6 [1, 9]; P < 0.001) between isoline and HART 89M.
Isoline was more susceptible to FOV and APH + FOV
than Bt cotton and HART 89M as shown by foliar symptoms and vascular discoloration (Fig. 1, Fig. 2). A significant treatment×treatment level interaction was observed
in the number of CFU collected from soil (F = 539.7 [1, 9];
186
Journal of Plant Pathology (2014), 96 (1), 183-188
Interaction of F. oxysporum and A. avenae
Table 1. Effect of Fusarium oxysporum f. sp. vasinfectum (FOV) and combined infections of FOV and Aphelenchus avenae
(APH + FOV) on plant growth parameters and colony forming units (CFU) of FOV in soil and stems of Bt cotton and its isoline.
Cultivar
Thesis
Plant height
No. of
nodes
No. of
bolls
Fresh shoot
weight
Fresh root
weight
CFU/g of
soil
CFU/g of
stem
No. of
nematodes
Bt cotton
CONTROL
FOV
APH
FOV+APH
CONTROL
FOV
APH
FOV+APH
57.3e
39.5b
48.4c
51.6d
63.6g
34.6a
57.8ef
58.5f
0.4
10.4d
8.4a
10.0c
9.5b
16g
11.4e
14.4f
15.9g
0.08
19.0g
12.7b
17.9f
17.5e
16.5d
12a
16.0c
15.9c
0.09
74.8e
46.5b
55.4c
72.0e
60.0d
33.9a
47.1b
54.5c
1.04
14.3e
11.7c
14.0de
11.6c
13.2d
6.5a
10.9c
8.8b
0.3
NA
1653c
NA
1464b
NA
1668d
NA
1350a
3.9
NA
1516c
NA
1407b
NA
1553d
NA
1354a
6.1
NA
NA
256.0a
390.7b
NA
NA
267.0c
399.5b
2.07
Isoline
SEM
Means within the same column with the same letter are not different (P < 0.05) according to least significant difference test (LSD).
P < 0.001) and roots (F = 54.6 [1, 9]; P < 0.001) of Bt cotton
and isoline. There were more CFU recovered from soil and
roots in the FOV treatment level of isoline than in the Bt
cotton (Table 1).
Plant growth parameters across all treatments were affected in a similar manner. FOV and APH+FOV caused
a reduction in plant height, number of nodes, number of
bolls, fresh shoot and root weight but the decrease was
greater in the FOV treatment. There was also a higher
reduction in growth parameters in FOV treatment than
in APH. There was a significant treatment × treatment
level interaction for plant height (F = 123.9 [3, 21]; P < 0.001),
number of nodes (F = 189.4 [3, 21]; P < 0.001), number of
bolls (F = 32.6 [3, 21]; P < 0.001), fresh shoot (F = 7.1 [3, 21];
P = 0.002) and root (F = 77.4 [3, 21]; P < 0.001) weight. There
was a significant difference in nematode populations
at 180 dap between the treatment levels (F = 4185 [1, 9];
P < 0.001) with higher numbers of nematodes recorded in
APH + FOV treatment level in both Bt cotton and isoline
treatments (Table 1). However, the number of nematodes
in the Bt cotton and isoline APH + FOV treatment were
not significantly different.
When the isoline and HART 89M were compared
to determine any varietal effects on interaction of FOV
and APH, the isoline was more susceptible to FOV inoculations than HART 89M (Fig. 1, Fig. 2). There was
significant treatment×treatment level interaction in
the number of CFU collected from soil (F = 1300.7 [1, 9];
P < 0.001) and roots (F = 375.5 [1, 9]; P < 0.001) of isoline
and HART 89M. FOV CFU were more abundant in soil
than in roots in both treatments. There was a significant
treatment×treatment level interaction for plant height
(F = 52.8 [3, 21]; P < 0.001), number of nodes (F = 161.6 [3, 21];
P < 0.001), fresh shoot (F = 7.13 [3, 21]; P < 0.001) and root
(F = 5.72 [3, 21]; P = 0.005) weight between HART 89M
and isoline treatment levels. There was a reduction in all
growth parameters in the FOV and APH + FOV treatments. A greater decrease in growth parameters was recorded in FOV than in APH and APH + FOV in both
cotton cultivars. A significant treatment×treatment level
interaction (F = 8.85 [1, 9]; P = 0.016) was observed in the
number of nematodes at 180 dap in HART 89M and
isoline treatment. A higher number of nematodes was recorded in APH + FOV in both HART 89M and isoline
treatment (Table 2). Bt protein was present in roots and
soil of Bt cotton at 180DAP in both trials. No Bt protein
was detected in HART 89M and isoline roots and soil.
DISCUSSION
Fungal-feeding nematodes thrive on beneficial and
pathogenic fungi and they are used as biological control agents (Freckman and Caswell, 1985). Pathogenic F.
oxysporum species cause vascular wilt and plant death in
many crops. They gain entry through the roots and invade
the vascular system from where they spread throughout
the plant.
In the present study, the main symptoms of fusarium
wilt, i.e. vascular browning and wilting were observed in
the treatment inoculated with FOV and APH+FOV. However, the isoline was more susceptible to fusarium wilt in
the single and combined infections than Bt cotton, an indication that Cry1Ac and Cry2Ab2 protein did not aggravate
disease severity or negatively affected the interaction of
FOV with A. avenae.
Bt protein was present in roots and soil at the end of
the growing period, as previously reported by Knox et
al. (2007). The presence of Bt protein has been shown to
cause differences in the response of Bt cotton to F. oxysporum. Resistance of plants to fusarium wilt is related to the
composition of root exudates and the presence of other
microorganisms. Disease-resistant cultivars provide few
nutrients to F. oxysporum or inhibit its growth by producing inhibitory compounds (Claudius and Mehrotra, 1972).
Unlike the low disease severity observed in this study, Li et
al. (2009) reported that two Bt cotton lines were more susceptible to F. oxysporum and their root exudates activated
mycelium growth and fungal spore germination. They also
reported differences in amino acids and sugars in the roots
of Bt cotton and non-Bt cotton and a high correlation of
disease indices with sugars.
Journal of Plant Pathology (2014), 96 (1), 183-188
Karuri et al. 187
Table 2. Effect of Fusarium oxysporum f. sp. vasinfectum (FOV) and combined infections of FOV and Aphelenchus avenae
(APH + FOV) on plant growth parameters and colony forming units (CFU) of FOV in soil and stems of isoline and HART 89M.
Cultivar
Thesis
Plant height
No. of
nodes
No. of bolls
Fresh shoot
weight
Fresh root
weight
CFU/g of
soil
CFU/g of
plant
No. of
nematodes
Isoline
CONTROL
FOV
APH
FOV+APH
CONTROL
FOV
APH
FOV+APH
63.5d
34.6a
57.8c
58.6c
92.7f
53.7b
85.1e
85e
0.43
16.0e
11.4a
14.4c
15.9e
16.6f
13.1b
15.5d
15.5d
0.05
16.5d
12.0a
16.0c
15.9c
12.5e
8.2b
12.4ef
12.2af
0.11
60e
33.9a
47.1c
54.5d
58.1e
37.1b
45.9c
59e
2.48
13.2d
6.6a
11c
8.9b
24.2g
12.9d
19.6f
17.5e
0.56
NA
1668c
NA
1350a
NA
1664c
NA
1474b
1.77
NA
1553c
NA
1354a
NA
1622d
NA
1514b
2.33
NA
NA
267.0b
399.5c
NA
NA
252.2a
406.6c
3.67
HART 89M
SEM
Means within the same column with the same letter are not different (P < 0.05) according to least significant difference test (LSD).
Minimal or no effects of Bt plants on fungal populations have been registered in different cases. For example:
(i) small differences were observed in the fungal populations of soils planted with transgenic Bt potato (Donegan
et al., 1995); (ii) the number of fungi did not differ in the
rhizosphere soil of Bt and non-Bt maize (Saxena and Stotzky, 2001b); (iii) Fusarium graminearum, and Trichoderma
atroviride were not affected by Cry1Ab protein in maize
(Naef et al., 2006). In this study, nematode populations
increased in APH+FOV treatment of Bt cotton and isoline, and but there was no significant difference in the
nematode numbers in the two treatments suggesting that
the interaction between the nematodes and fungi was not
affected by Bt protein. The number of A. avenae were
higher in the nematodes and fungi combination than in
the nematode alone treatment, which may be due to the
fact that F. oxysporum is among the preferred food sources
of APH (Chen and Ferris, 2000).
Although the isoline was more susceptible to fusarium
wilt than the other treatments, the disease caused a reduction in growth parameters in all treatments of APH + FOV
and FOV levels. Plant growth variables including weight,
height and number of nodes were negatively affected by
infection with FOV alone and APH + FOV. The numbers
of bolls in the FOV treatment were fewer than those in
APH + FOV. Devay et al. (1997) reported that vascular
browning in cotton was associated with a decrease in plant
growth and development of nodes. Hao et al. (2009) also
reported a negative correlation between cotton growth
variables and FOV. Disease severity was much less in the
APH+FOV treatment compared to FOV, which is an indication of the ability of APH to suppress fusarium wilt in
cotton. Lagerlof et al. (2011) demonstrated that this nematode could suppress damping off in cauliflower seedlings
caused by Rhizoctonia solani.
Damping off of radish caused by Pythium spp. was also
shown to be controlled by APH (Jun and Kim, 2004),
while Barnes et al. (1981) showed that Rhizoctonia solani
and F. solani could be controlled by the same nematode.
Hussey and Roncadori (1981) reported that pathogenicity
of some root rotting fungi was reduced by fungal-feeding
nematodes. Despite the fact that no effect of Bt cotton on
trophic interactions between APH and FOV was observed
in the greenhouse, this may not be reflected in the field
since other environmental factors can affect the activities
of these organisms and influence the contribution of each
in the interactions. Similar studies should be carried out
under field conditions to confirm the greenhouse results.
ACKNOWLEDGEMENTS
This is paper No 31 of the BiosafeTrain project funded by the Danish International Development Agency
(DANIDA). The authors thank the Kenya Agricultural
Research Institute, National Agricultural Research Laboratories (KARI-NARL) for providing laboratory space. We
acknowledge Mette Vesteergard for assistance in nematode
identification, Maurice Okomo and Mary Ndunguli for
technical support and Elias Thuranira for assistance in
data analysis.
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