Arbuscular mycorrhizal fungi induce systemic resistance in tomato against the sedentary nematode Meloidogyne incognita and the migratory nematode Pratylenchus penetrans

Applied Soil Ecology 61 (2012) 1–6

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Arbuscular mycorrhizal fungi induce systemic resistance in tomato against the sedentary nematode Meloidogyne incognita and the migratory nematode Pratylenchus penetrans C.M. Vos a,∗ , A.N. Tesfahun a , B. Panis a , D. De Waele a,c , A. Elsen b,c a Laboratory of Tropical Crop Improvement, Department of Biosystems, Faculty of Bioscience Engineering, University of Leuven (K.U.Leuven), Kasteelpark Arenberg 13, B-3001 Leuven, Belgium b Bodemkundige Dienst van België, Willem de Croylaan 48, 3001 Heverlee, Belgium c Department of Biology, Faculty of Sciences, Ghent University, Ledeganckstraat 35, B-9000 Gent, Belgium

a r t i c l e

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Article history: Received 23 November 2011 Received in revised form 18 April 2012 Accepted 24 April 2012 Keywords: Biological control Glomus mosseae Meloidogyne incognita Pratylenchus penetrans Split-root Tomato

a b s t r a c t Arbuscular mycorrhizal fungi (AMF) are able to protect plants against a range of soil-borne pathogens, but the biocontrol modes of action remain largely unknown, especially in the case of plant-parasitic nematodes. As a first step toward the elucidation of the modes of action, the interaction between the AMF Glomus mosseae and two nematode species with a different infection cycle was studied in tomato: the sedentary root-knot nematode Meloidogyne incognita and the migratory root-lesion nematode Pratylenchus penetrans. In greenhouse interaction experiments, nematode infection was investigated 8 weeks (M. incognita) or 10 weeks (P. penetrans) after nematode inoculation, in tomato roots either colonized by G. mosseae or not. The mycorrhiza-induced resistance was confirmed against both nematode species as the nematode population was significantly lower in mycorrhizal roots, with an overall reduction of 45% in the case of M. incognita and 87% for P. penetrans, although the latter nematode showed low infection numbers in tomato. In subsequently conducted split-root experiments, consisting of a two-compartment set-up, AMF were applied either locally (i.e. nematodes in the same compartment as AMF) or systemically (i.e. nematodes and AMF physically separated). The presence of AMF, either local or systemic, resulted in a significant infection reduction for both nematodes. The results show for the first time that infection by the root-knot nematode M. incognita and the root-lesion nematode P. penetrans is systemically reduced by G. mosseae. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Root-knot nematodes (Meloidogyne spp.) and root-lesion nematodes (Pratylenchus spp.) are worldwide important pathogens of a wide range of agricultural crops. Both types of nematodes are endoparasitic, since they completely enter the plant roots, but display a very different feeding behavior inside the roots. Sedentary nematodes, like Meloidogyne incognita, induce and maintain specialized feeding cells, so-called giant cells, around their immobile body in the central cylinder of the root. Hyperplasia and hypertrophy of the surrounding cells lead to the formation of typical root galls. The 2nd stage juveniles are the only infective, mobile life stage of the nematode (Caillaud et al., 2008). For migratory nematodes on the other hand, like Pratylenchus penetrans, all life stages are able to migrate through the root cortex while feeding on ever new cortical cells, which leads to extensive root necrosis (Zunke, 1990).

∗ Corresponding author. Tel.: +32 16 32 96 03; fax: +32 16 321993. E-mail address: [email protected] (C.M. Vos). 0929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsoil.2012.04.007

The damage potential of root-knot nematodes can be extremely high and reach up to 100% in various crops (overview in Wesemael et al., 2011), while the root-lesion nematodes rank second only to them with respect to yield loss (Moens and Perry, 2009). Traditionally, nematode infection is managed by using nematicides, but growing concern arises about their environmental impact. For example, the most effective and widely used fumigant, methyl bromide, has already been banned since 2005 and the use of other fumigant pesticides in Europe is restricted (Dong and Zhang, 2006; Wesemael et al., 2011). These decisions have stimulated research for alternative control practices, including the use of biological control organisms. Arbuscular mycorrhizal fungi (AMF) have also come into the picture as biological control organisms. AMF are obligate root symbionts of 80% of the plants that can improve plant growth through increased nutrient uptake and also alleviate plant stress caused by abiotic and biotic factors, in exchange for photosynthetic carbon from their host. In addition, AMF provide other ecosystem services such as increased soil stability and reduction of fertilizer requirements (Smith and Read, 2008; Gianinazzi et al., 2010). The

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biocontrol effect of AMF has been observed in a wide range of plants against different pathogens, as reviewed by Whipps (2004) and Pozo and Azcon-Aguilar (2007). Most reports deal with soilborne fungal pathogens, but the interaction with some bacterial and aboveground pathogens has also been investigated. Despite the numerous reports on the biocontrol effect of AMF, their actual use as biological control agents is still limited, partially due to variability in performance, depending on the AMF isolate, pathogen, plant and environmental conditions (Dong and Zhang, 2006). A better insight into the biocontrol modes of action can help to fine-tune the efficacy of this biocontrol agent. Four categories of possible modes of action were distinguished by Whipps (2004); these include (i) direct competition or inhibition; (ii) enhanced or altered plant growth, morphology and nutrition; (iii) biochemical changes associated with plant defense mechanisms and induced resistance, and (iv) development of an antagonistic microbiota. Biocontrol effects against nematodes in the local presence of various AMF species have been reported in a wide array of plants, mainly for the genera Heterodera, Meloidogyne, Pratylenchus and Radopholus. However, the modes of action remain speculative (reviews by Hol and Cook, 2005; Akhtar and Siddiqui, 2008). Systemic resistance induced by AMF has been shown in interactions with several pathogens (Cordier et al., 1998; Pozo et al., 2002; Zhu and Yao, 2004; Fritz et al., 2006; Khaosaad et al., 2007; Elsen et al., 2008; Castellanos-Morales et al., 2011) and might also be reflected in the phenomenon of systemic autoregulation of the mycorrhizal colonization (Pinior et al., 1999; Vierheilig et al., 2003). It has been hypothesized that plants use the autoregulation mechanism as a preventative measure against further mycorrhizal colonization and as a defense against pathogens at the same time (Vierheilig and Piché, 2002; Vierheilig et al., 2008). The mycorrhiza-induced resistance shares similarities with rhizobacteria-mediated induced systemic resistance (ISR): both defense mechanisms seem to be based on priming of jasmonateregulated responses (reviewed by Pozo and Azcon-Aguilar, 2007), the resistance does not depend on accumulation of salicylic acid (Khaosaad et al., 2007), and similar structural modifications at the cytological level have been reported in both cases (Cordier et al., 1998). Although several reports demonstrate the biocontrol potential of various species of AMF against several plant-parasitic nematode species in a wide range of plants and experimental conditions, the modes of action involved still remain unclear. Investigating whether the biocontrol potential of AMF against nematodes is systemic, forms a first step toward more insight into the modes of action. Observation of such a systemic effect would imply the involvement of a signal that is transferred throughout the host plant and would then merit further in-depth analysis of the plant response. So far, the systemic nature of the mycorrhiza-induced resistance toward nematodes had only been observed once up to now, in the case of the migratory burrowing nematode Radopholus similis in banana colonized by Glomus intraradices (Elsen et al., 2008). Therefore, the objective of the present study was to determine whether the commonly used AMF Glomus mosseae is able to induce systemic resistance in tomato plants against the economically important endoparasitic sedentary root-knot nematode M. incognita and the migratory root-lesion nematode P. penetrans.

2. Materials and methods 2.1. Biological materials In all experiments, the tomato (Lycopersicon esculentum) cultivar Marmande was grown under greenhouse conditions at an ambient

temperature of 20–27 ◦ C and 75% relative humidity. The AMF G. mosseae, previously isolated from banana in Tenerife, Spain, and maintained as a greenhouse stock culture on sorghum, was applied as a layer at sowing, between two layers of substrate. The inoculum consisted of rhizosphere soil from 6-month old sorghum pot cultures containing spores, hyphae and heavily colonized root pieces. Plants from the non-mycorrhizal treatment received rhizosphere soil from non-colonized sorghum plants. At the end of every experiment, plants from the non-mycorrhizal treatment were randomly selected and tested for mycorrhizal colonization. Mycorrhizal colonization was determined by staining the roots with an ink-vinegar solution (Vierheilig et al., 1998). After clearing in 10% KOH, staining in 5% ink-vinegar solution and destaining in water, 20 root pieces of 1 cm were mounted on slides and observed with a light microscope. The frequency of mycorrhizal colonization (F%) was calculated as the percentage of root segments colonized by hyphae, arbuscules or vesicles. In addition, the abundance of these structures, i.e. the intensity of the mycorrhizal colonization (I%), was estimated visually using different classes (1–20, 20–40, 40–60, 60–80, 80–100%) that correspond to the proportion of the cortex colonized by the mycorrhizal fungus. The results were expressed as a percentage by calculating (20x + 40y + 60z + 80t + 100v)/(x + y + z + t + v) with x, y, z, t and v the number of root fragments in each class (Plenchette and Morel, 1996). The M. incognita population was originally isolated from banana in Malaysia and maintained as a greenhouse stock culture on tomato cv. Marmande. A pure culture was initiated in the greenhouse starting from a single egg mass. Inoculum was obtained after four months by extraction of egg masses from heavily infected tomato roots, followed by 5-day incubation on modified Baermann dishes to obtain freshly hatched 2nd stage juveniles (Hooper et al., 2005). The P. penetrans population was originally isolated from potato in Belgium and monoxenically cultured in vitro on carrot discs at 21 ◦ C in the dark (Pinochet et al., 1995). A pure culture was initiated on carrot discs starting from a single female. After ten weeks, the inoculum was extracted from carrot discs using the maceration-sieving method (Speijer and De Waele, 1997). 2.2. Interaction experiments Tomato seeds were planted in the greenhouse with the same climatic conditions as mentioned above, in 1 l pots containing a substrate mixture of sand and potting soil (2:1). For the mycorrhizal treatment, 200 ml of rhizosphere soil colonized by G. mosseae was added to each pot, as described before. Plants from the nonmycorrhizal treatment received 200 ml of rhizosphere soil from non-colonized sorghum plants. All plants were placed in a completely randomized design and each treatment consisted of 8 replications. After 6 weeks, 8 mycorrhizal plants were uprooted to determine mycorrhizal colonization as described before. After determination of mycorrhizal colonization, plants were inoculated with either M. incognita or P. penetrans, while 8 mycorrhizal plants were not inoculated with nematodes to assess the mycorrhizal colonization at the end of the experiment. The M. incognita inoculum consisted of 3000 freshly hatched 2nd stage juveniles per plant. Pots were watered to field capacity at time of inoculation, and 3000 nematodes were inoculated in 4 ml of water into two 1-cm deep holes in the substrate around the stem base. Eight weeks after inoculation, plants were uprooted and root galling was estimated according to the diagrammatic root-knot scoring chart by Bridge and Page (1980). Severity of root galling is rated on a scale from 1 (few small knots, difficult to find) to 10 (all roots severely knotted, no root system, plant usually dead). Nematode infection was assessed by gently washing the roots, followed by immersing a 2 g root subsample in 0.015% Phloxine-B for 15 min to stain the egg masses and count the corresponding egg

C.M. Vos et al. / Applied Soil Ecology 61 (2012) 1–6

laying females (Hooper et al., 2005). Nematodes were extracted from the second 2 g subsample by maceration-sieving (Speijer and De Waele, 1997). Roots were cut into 1-cm segments and macerated in a 1% solution of NaOCl in a blender for two times 10 s. The homogenized solution was then passed through a series of sieves (250–100–25 ␮m sieve) and the collected 2nd stage juveniles and eggs were counted with a light microscope. The P. penetrans inoculum consisted of 1000 vermiform nematodes (71% juveniles, 18% females, 11% males) per plant, and nematodes were inoculated in 2 ml of water into two 1-cm deep holes in the substrate around the stem base. Ten weeks after inoculation, the percentage of root necrosis was measured by scoring five 10-cm functional roots (Speijer and De Waele, 1997), and the number of nematodes inside the roots was determined by maceration of the roots in distilled water for two times 10 s, followed by sieving of the homogenized solution (250–100–25 ␮m sieve) (Speijer and De Waele, 1997). Nematodes were collected from the 25 ␮m sieve and the number of juveniles, females and males was counted using light microscopy. The reproduction factor (Rf ) for P. penetrans was calculated as the final nematode population (Pf ) divided by the inoculated initial nematode population (Pi ).

2.3. Split-root experiments For the split-root experiments, tomato seeds were planted in seeding trays in the greenhouse under the climatic conditions mentioned above. Three weeks after sowing, enough roots had developed and the plants were transferred to the split-root set-up, consisting of two 200 ml compartments connected in the middle. Both sides of the split-root set-up were filled with a mixture of sand and potting soil (2:1). For the mycorrhizal treatments, 40 ml of rhizosphere mycorrhizal inoculum was added as a layer, as described before. Plants from the non-mycorrhizal treatment received 40 ml of rhizosphere soil from non-colonized sorghum plants. All plants were placed in a completely randomized design and each treatment consisted of 8 replications. The tomato seedlings were placed on top of the split-root set-up and their root systems divided over the two compartments. The plants were kept for 6 weeks to allow sufficient root development in both compartments and mycorrhizal colonization in the mycorrhizal compartments. After 6 weeks, mycorrhizal colonization was determined and the right compartment of the split-root was inoculated with 200 freshly hatched M. incognita 2nd stage juveniles per plant or 200 vermiform P. penetrans nematodes (56% juveniles, 32% females, 12% males) per plant, while leaving the left compartment uninoculated. After 8 (for M. incognita) or 10 weeks (for P. penetrans), the plants were harvested and the mycorrhizal colonization and nematode infection were determined in both compartments in the same way as described above. Three treatments were included in each experiment: control (i.e. nematodes in the right compartment), local AMF (i.e. AMF and nematodes together in the right compartment) and systemic AMF (i.e. AMF in the left compartment and nematodes in the right compartment).

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3. Results A well-established and active mycorrhizal colonization was observed in both interaction experiments, as hyphal structures, arbuscules and vesicles were all present in the stained roots. Colonization frequency was close to 100% in both experiments, with an average colonization intensity of 33% prior to nematode inoculation. Nematode infection with either M. incognita or P. penetrans did not affect mycorrhizal colonization of the tomato roots as indicated by the colonization parameters observed 8 weeks (for M. incognita) or 10 weeks (for P. penetrans) after nematode inoculation (Table 1). No mycorrhizal colonization was observed in the randomly selected plant roots of the non-mycorrhizal treatment. Plant root and shoot weight did not differ significantly between mycorrhizal and non-mycorrhizal plants (Tables 2 and 3). In the interaction experiments, mycorrhizal colonization significantly reduced the infection of M. incognita in tomato roots, as indicated by the significantly lower number of 2nd stage juveniles (46% reduction), egg-laying females (34% reduction) and eggs (45% reduction) in mycorrhizal roots (Table 2). Mycorrhizal plants had an average gall index of 4 (larger knots predominate but main roots clean), while the gall index of non-mycorrhizal plants was significantly higher, with an average index of 7 (majority of main roots knotted) (Table 2). For P. penetrans, significantly less females were observed in the mycorrhizal roots compared to the nonmycorrhizal roots, and the overall reproduction rate of P. penetrans was significantly lower in mycorrhizal roots, being only 13% of that in the non-mycorrhizal roots (Table 3). Reproduction of P. penetrans on tomato was low, as indicated by a reproduction rate lower than 1 (Table 3). Root necrosis varied between 0 and 4% in both treatments and did not differ significantly between the treatments. In the split-root experiments, mycorrhizal colonization in the AMF compartments reached an average frequency of 78% and intensity of 37%, with observations of hyphal structures, arbuscules as well as vesicles. No mycorrhizal structures were detected in the non-mycorrhizal compartments of the split-root set-up. Mycorrhizal colonization did not significantly alter either root weight or shoot weight of the plants (Tables 4 and 5). Nematode infection in the split-root experiments did not spread to the non-inoculated compartments. Significantly less M. incognita 2nd stage juveniles were extracted from roots in the presence of AMF, either local (71% reduction) or systemic (68% reduction), but the reduction in the number of egg-laying females was not significant (Table 4). Significantly less eggs were found in the local AMF (47% reduction) and systemic AMF treatment (43% reduction) compared to non-mycorrhizal roots. Mycorrhizal plants in both the local AMF and systemic AMF treatment had an average gall index of 3 (some larger knots visible, main roots clean), while the gall index of control plants averaged 5 (50% of roots affected, knotting on some main roots, reduced root system) (Table 4). In the case of P. penetrans, significantly less juveniles were observed both in the local AMF and the systemic AMF treatment compared to the non-mycorrhizal treatment, with a reduction of 60% and 51% respectively. A similar trend could be observed for the reproduction rate. Root necrosis varied between 0 and 2% in all treatments and did not differ significantly (Table 5).

2.4. Statistical analysis Nematode infection, mycorrhizal colonization, and plant data of all experiments were statistically analyzed by analysis of variance (ANOVA) when the conditions for ANOVA were met (i.e. normal distribution and homogeneity of variances) using Statistica® software (Release 7, Statsoft). The Tukey HSD test was applied for multiple comparisons of group means. Prior to analysis, nematode numbers were log(x + 1) transformed, and percentages were arcsin(x/100) transformed, to reduce data variance.

4. Discussion Both in the interaction experiments and split-root experiments a biocontrol effect was demonstrated against the sedentary M. incognita and the migratory P. penetrans in tomato colonized by G. mosseae. This is in accordance with earlier reports about the mycorrhiza-induced resistance against endoparasitic nematodes in various plant species, as reviewed by Hol and Cook (2005) and

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Table 1 Frequency (F%) and intensity (I%) of Glomus mosseae colonization of tomato roots, just before nematode inoculation (0 WAI) and 8 weeks (8 WAI, for M. incognita) or 10 weeks (10 WAI, for P. penetrans) after inoculation. Control plants were not inoculated with nematodes. Time

M. incognita

0 WAI 8 or 10 WAI P (time)

P. penetrans

Control

F%

I%

F%

I%

F%

I%

98 ± 2 96 ± 1 n.s.

33 ± 5 37 ± 5 n.s.

98 ± 2 99 ± 1 n.s.

33 ± 5 36 ± 4 n.s.

98 ± 2 97 ± 2 n.s.

33 ± 5 38 ± 4 n.s.

Data represent mean ± standard error of 8 replications. No significant differences (P ≤ 0.05) were observed according to one-way ANOVA and Tukey’s HSD test. Statistical analysis was performed on arcsin(x/100) transformed data. n.s., not significant.

Table 2 Plant growth parameters and nematode infection data of tomato either non-colonized (− AMF) or colonized by Glomus mosseae (+ AMF), 8 weeks after inoculation with Meloidogyne incognita (Pi = 3000 2nd stage juveniles). Treatment

RW (g)

SW (g)

Juvenilesa

Females

Males

Eggs

Gall indexb

− AMF + AMF P (AMF)

31 ± 4 a 28 ± 2 a n.s.

44 ± 5 a 50 ± 3 a n.s.

28 387 ± 3884 b 15 431 ± 1938 a 0.006

817 ± 99 b 536 ± 70 a 0.045

– – n.a.

112 046 ± 16 295 b 61 195 ± 6927 a 0.018

7 ± <1 b 4 ± <1 a 0.016

Data represent mean ± standard error of 8 replications. Within each column, values followed by different letters are significantly different (P ≤ 0.05) according to one-way ANOVA and Tukey’s HSD test. Statistical analysis was performed on log(x + 1) transformed nematode infection data. n.s., not significant; n.a., not applicable; SW, shoot weight; RW, root weight. a The number of M. incognita juveniles refers to 2nd stage juveniles. b Gall index was rated on a scale of 1–10, according to Bridge and Page (1980). Table 3 Plant growth parameters and nematode infection data of tomato either non-colonized (− AMF) or colonized by Glomus mosseae (+ AMF), 10 weeks after inoculation with Pratylenchus penetrans (Pi = 1000 vermiform nematodes). Treatment

RW (g)

SW (g)

Juveniles

Females

Males

Rf

Root necrosisa (%)

− AMF + AMF P (AMF)

27 ± 3 a 22 ± 2 a n.s.

61 ± 2 a 57 ± 3 a n.s.

324 ± 144 a 88 ± 35 a n.s.

250 ± 84 b <1 ± <1 a 0.002

94 ± 67 a <1 ± <1 a n.s.

0.7 ± 0.2 b 0.1 ± 0.1 a 0.036

2±2 a 2±2 a n.s.

Data represent mean ± standard error of 8 replications. Within each column, values followed by different letters are significantly different (P ≤ 0.05) according to one-way ANOVA and Tukey’s HSD test. Statistical analysis was performed on log(x + 1) or arcsin(x/100) transformed data. n.s., not significant. Reproduction factor Rf = final nematode population (Pf )/initial nematode population (Pi ). SW, shoot weight; RW, root weight. a Percentage of root necrosis was scored according to Speijer and De Waele (1997). Table 4 Plant growth parameters and nematode infection data of tomato in the right compartment of the split-root set-up, either non-colonized or colonized by Glomus mosseae, 8 weeks after inoculation with Meloidogyne incognita (Pi = 200 2nd stage juveniles). Treatment

RWa (g)

SW (g)

Juvenilesb

Females

Males

Eggs

Gall indexc

Control Local AMF Systemic AMF P (treatment)

11 ± 2 a 8±1 a 10 ± 2 a n.s.

47 ± 4 a 52 ± 3 a 54 ± 3 a n.s.

2165 ± 223 b 626 ± 186 a 682 ± 148 a 0.007

334 ± 66 a 133 ± 48 a 179 ± 47 a 0.053

– – – n.a.

7265 ± 1103 b 3853 ± 630 a 4120 ± 752 a 0.041

5 ± <1 b 3 ± <1 a 3 ± <1 a 0.032

Data represent mean ± standard error, based on 8 replications. Within each column, values followed by different letters are significantly different (P ≤ 0.05) according to one-way ANOVA and Tukey’s HSD test. Statistical analysis was performed on log(x + 1) transformed data. n.s., not significant; n.a., not applicable. Control treatment (i.e. nematodes in right compartment of split root), local AMF treatment (i.e. AMF and nematodes in right compartment of split root), systemic AMF treatment (i.e. AMF in left compartment and nematodes in right compartment of split root). a Root weight data refer to the right compartment of the split-root. b The number of M. incognita juveniles refers to 2nd stage juveniles. c Gall index was rated on a scale of 1–10, according to Bridge and Page (1980). Table 5 Plant growth parameters and nematode infection data in tomato in the right compartment of the split-root set-up, either non-colonized or colonized by Glomus mosseae, 10 weeks after inoculation with Pratylenchus penetrans (Pi = 200 vermiform nematodes). Treatment

RWa (g)

SW (g)

Juveniles

Females

Males

Rf

Root necrosisb (%)

Control Local AMF Systemic AMF P (treatment)

7±2 a 10 ± 2 a 8±1 a n.s.

51 ± 2 a 56 ± 3 a 55 ± 3 a n.s.

43 ± 10 b 17 ± 4 a 21 ± 5 a 0.037

10 ± 3 a 8±3 a 8±3 a n.s.

3±1 a 8±4 a 2±1 a n.s.

0.28 ± 0.06 b 0.16 ± 0.05 a 0.16 ± 0.04 a 0.046

1 ± <1 a <1 ± <1 a 1 ± <1 a n.s.

Data represent mean ± standard error, based on 8 replications. Within each column, values followed by different letters are significantly different (P ≤ 0.05) according to oneway ANOVA and Tukey’s HSD test. Statistical analysis was performed on log(x + 1) or arcsin(x/100) transformed data. n.s., not significant. Control treatment (i.e. nematodes in right compartment of split root), local AMF treatment (i.e. AMF and nematodes in right compartment of split root), systemic AMF treatment (i.e. AMF in left compartment and nematodes in right compartment of split root). Reproduction factor Rf = final nematode population (Pf )/initial nematode population (Pi ). a Root weight data refer to the right compartment of the split-root. b Percentage of root necrosis was scored according to Speijer and De Waele (1997).

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Akhtar and Siddiqui (2008). However, the same reviews made it also clear that the biocontrol effect can be less effective or even absent, depending on AMF isolate, plant species, pathogen species, soil and other environmental conditions (Whipps, 2004). One established prerequisite is the degree of mycorrhizal colonization, a condition that was met in the experiments presented in this study. Pozo and Azcon-Aguilar (2007) stressed the importance of such a mature symbiosis, characterized by the presence of arbuscules, as a prerequisite for biocontrol. This was also observed by Slezack et al. (2000) who reported that G. mosseae-induced biocontrol only took place when the AMF could establish a fully functional symbiosis with their host. Apart from the observation on the importance of a mature symbiosis, however, so far few attempts have been made toward explaining the modes of action of the observed mycorrhiza-induced resistance against plant-parasitic nematodes. In this study we demonstrate the systemic nature of the mycorrhiza-induced resistance against the sedentary M. incognita and the migratory P. penetrans. This observation implies that G. mosseae is able to exert an indirect effect on the nematodes through modification of the host plant. This merits further investigation that can lead to more insight into the modes of action of the mycorrhiza-induced resistance. The systemic nature of the mycorrhiza-induced resistance toward nematodes had only been observed once up to now, in the case of the migratory burrowing nematode R. similis in banana colonized by G. intraradices (Elsen et al., 2008). In another monocotyledonous plant, the dunegrass Ammophila arenaria, no systemic resistance against P. penetrans was observed after colonization by ˜ et al., 2006). Moreover, a systemically native AMF (De la Pena induced mycorrhizal biocontrol effect for sedentary nematodes had not yet been reported to our knowledge. Our findings shed some light on the plausibility of some of the modes of action proposed for mycorrhiza-induced resistance (Whipps, 2004). Since we observed a biocontrol effect when AMF and nematodes were physically separated, a direct competition between the two organisms is not possible. In addition, growth promotion of tomato colonized by G. mosseae was not observed in our experiments, which corresponds with the generally low response of tomato toward AMF in terms of plant growth, as reviewed by Smith et al. (2009). Mycorrhizal colonization indeed does not always result in growth promotion, since the fungus represents a carbon cost for the plant that cannot be compensated in some cases. The net result on plant growth can vary depending on the selected AMF, the plant cultivar, and on other factors such as soil chemistry (Smith and Smith, 2011). As such, the hypothesis that mycorrhizal plants can overcome pathogen infections by increased plant growth is not supported. As far as the molecular basis of the mycorrhiza-induced resistance against plant-parasitic nematodes is concerned, reports are scarce. Li et al. (2006) reported the primed transcriptional activation of a class III chitinase gene in mycorrhizal grapevine roots upon infection by M. incognita. Thus far, research regarding the modes of action of mycorrhizainduced resistance has focused mainly on the interaction with pathogenic fungi and to some extent on the interaction with pathogenic bacteria. By applying the split-root set-up, several authors have demonstrated the systemic induced resistance in mycorrhizal plants toward different fungal or bacterial soil-borne pathogens (Cordier et al., 1998; Pozo et al., 2002; Zhu and Yao, 2004; Khaosaad et al., 2007). But mycorrhiza-induced systemic resistance does not necessarily seem to be confined to the root. Extension toward the aerial plant parts seems possible as well since Fritz et al. (2006) reported that AMF conferred systemic resistance toward the leaf pathogen Alternaria solani in tomato. In most cases, however, the resulting net biocontrol effect is reported to be the outcome of interplay between local and systemic plant defense

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responses. In tomato roots infected with Phytophthora parasitica, the plant cells containing AMF were reinforced by callose, while the systemic response involved elicitation of host wall thickenings in addition to callose-rich encasement material around the penetrating hyphae of the fungal pathogen (Cordier et al., 1998). Pozo et al. (2002) observed the induction of AMF-related new isoforms of the hydrolytic enzymes chitinase, chitosanase and ␤-1,3-glucanase in the same three-way interaction to be confined to mycorrhizal root tissue alone, while lytic activity against the cell wall of the pathogen seemed also systemically induced. The authors also reported different enzyme patterns depending on the AMF species, either G. mosseae or G. intraradices, indicating the complexity and specificity of the systemic resistance induced by AMF. For nematodes, the molecular basis of the mycorrhiza-induced systemic resistance still needs to be unraveled. The involvement of ISR in nematode infection induced by other organisms than AMF remains a largely unknown research area as well. The presence of certain rhizospheric bacteria like Pseudomonas spp. led to ISR against the sedentary cyst nematode Globodera pallida in potato (Hasky-Günther et al., 1998) and against the root-knot nematodes M. incognita (Munif et al., 2001) and Meloidogyne javanica (Siddiqui and Shaukat, 2002) in tomato. ISR was also observed with preinoculation of Fusarium oxysporum endophytes in banana against the migratory burrowing nematode R. similis (Vu et al., 2006). In conclusion, the presented data confirm the biocontrol potential of the AMF G. mosseae against two different types of endoparasitic nematodes and report for the first time that, similar to ISR, infection by the root-knot nematode M. incognita is systemically reduced by AMF. In view of the different infection cycles of sedentary and migratory nematodes, it would not be surprising if different mechanisms are responsible for the same effect, as Whipps (2004) already suggested in the case of other pathogens. Concerning in-depth investigations of biological control interactions, the nematology field lags behind. The local and systemic plant defense responses leading toward mycorrhiza-induced resistance as well as the signaling pathways involved remain unknown in the case of nematode infection and deserve further in-depth investigation. As already mentioned by Dong and Zhang (2006), a better understanding of the complex interaction will allow the fine-tuning of biological control of plant-parasitic nematodes and increase the efficacy of potential biological control agents in field applications. Acknowledgments This work was supported by a specialization grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) to C. Vos and a VLIR-UDC grant from the Belgian Government to A.N. Tesfahun. References Akhtar, M.S., Siddiqui, Z.A., 2008. Arbuscular mycorrhizal fungi as potential bioprotectants against plant pathogens. In: Siddiqui, Z.A., Akhtar, M.S., Futai, K. (Eds.), Mycorrhizae: Sustainable Agriculture and Forestry. Springer Science + Business Media B.V, pp. 61–97. Bridge, J., Page, S.L.J., 1980. Estimation of root-knot nematode infestation levels on roots using a rating chart. Trop. Pest Manag. 26, 296–298. Caillaud, M., Dubreuil, G., Quentin, M., Perfus-Barbeoch, L., Lecomte, P., de Almeida Engler, J., Abad, P., Rosso, M., Favery, B., 2008. Root-knot nematodes manipulate plant cell functions during a compatible interaction. J. Plant Physiol. 165, 104–113. Castellanos-Morales, V., Keiser, C., Cárdenas-Navarro, R., Grausgruber, H., Glauninger, J., García-Garrido, J.M., Steinkellner, S., Sampedro, I., Hage-Ahmed, K., Illana, A., Ocampo, J.A., Vierheilig, H., 2011. The bioprotective effect of AM root colonization against the soil-borne fungal pathogen Gaeumannomyces graminis var. tritici in barley depends on the barley variety. Soil Biol. Biochem. 43, 831–834. Cordier, C., Pozo, M.J., Barea, J.M., Gianinazzi, S., Gianinazzi-Pearson, V., 1998. Cell defense responses associated with localized and systemic resistance to

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