Fusarium confers protection against several ... - Wiley Online Library

Plant Pathology (2005) 54, 773–780

Doi: 10.1111/j.1365-3059.2005.01285.x

Fusarium confers protection against several mycelial pathogens of pepper plants

Blackwell Publishing, Ltd.

J. Díaz*†, C. Silvar, M. M. Varela‡, A. Bernal and F. Merino Depto. de Bioloxía Animal, Bioloxía Vexetal e Ecoloxía, Universidade da Coruña, Campus da Zapateira s/n, E−15071, A Coruña, Spain

Inoculation of nonhost pepper (Capsicum annuum) plants with the tomato wilt pathogen, Fusarium oxysporum f.sp. lycopersici (FOL), caused no symptoms and the fungus was not recovered from any part of the plant. FOL, however, partially protected pepper plants from subsequent infection with Phytophthora capsici, Verticillium dahliae or Botrytis cinerea by significantly reducing the percentage of diseased plants and the appearance and intensity of symptoms. FOL did not inhibit the mycelial growth of these pathogens in vitro. The protection induced by FOL against Botrytis was inhibited by 1-methylcyclopropene (MCP), an inhibitor of ethylene perception, suggesting the involvement of this hormone in the signalling of FOL-induced resistance. The activities of β-1,3-glucanase and peroxidase 48 h after FOL induction were similar to those in control plants. Chitinase activity, however, was higher in the stems of plants inoculated with FOL. A study of the levels of phenolic compounds revealed that cell-wall-bound phenolics were more abundant in plants treated with FOL, especially in stems, while soluble phenolic contents did not differ. Keywords: chitinase, ethylene, Fusarium oxysporum f.sp. lycopersici, induced systemic resistance, PR proteins

Introduction Pepper (Capsicum annuum) is cultivated in many areas of the world, including Spain, for its edible fruits. The crop is attacked by several pathogens, causing serious losses in production. Some of the most common are Phytophthora capsici (Hausbeck & Lamour, 2004), Verticillium dahliae (Bhat et al., 2003) and Botrytis cinerea (Mercier et al., 2001). The latter two pathogens are difficult to control by cultural methods or fungicides, and in the case of P. capsici, although phenylamides have been effective in disease control, resistant forms of the pathogen have appeared in the field (Parra & Ristaino, 2001; Hausbeck & Lamour, 2004). Moreover, the intensive and long-term use of fungicides in disease control can sometimes lead to serious soil contamination that should be avoided. There are pepper lines resistant to P. capsici (Oelke et al., 2003), but they are not always suitable for market purposes, particularly in Galicia (northwestern Spain), where the local market demands the Padron pepper, a low-pungency *To whom correspondence should be addressed. †E-mail: [email protected] ‡Present address: Instituto Español de Oceanografía, Centro Oceanográfico de A Coruña, Apdo. 130, E−15080, A Coruña, Spain. Accepted 18 May 2005

© 2005 BSPP

hot pepper consumed when immature (Estrada et al., 2000). There are no pepper lines fully resistant to V. dahliae, and resistance breeding has been only partially successful (Palloix et al., 1990; González-Salán & Bosland, 1991). Furthermore, no resistance to B. cinerea has been developed. One of the more promising strategies for controlling plant pathogens is the management of induced systemic resistance. This is a phenomenon in which resistance to infectious disease is systemically induced by localized infection by other microorganisms, by treatment with microbial components or products, or by a diverse group of structurally unrelated inorganic or organic compounds (KuÇ, 2000). One advantage of this strategy is that plants become immunized against a wide range of pathogens, even if the protection obtained is usually not complete. The number of studies on induced resistance has increased in past decades, and some forms of this resistance, namely systemic acquired resistance (SAR) and plant growth promoting rhizobacteria-induced systemic resistance (PGPR-ISR), have been relatively well characterized in their signalling pathways, the range of pathogens for which protection is acquired and, in the case of SAR, associated changes in expression of pathogenesis-related (PR) proteins (Pieterse & van Loon, 1999). A vast range of compounds and microorganisms has been assayed to obtain induced resistance. However, this is not the only way in which microorganisms can protect the plant from disease. Microorganisms can also affect pathogens by producing hydrolytic enzymes and antibiotics, 773

774

J. Díaz et al.

competing for nutrients and interfering with pathogenicity factors; some are already available as commercial products (Punja & Utkhede, 2003). The microorganisms used for inducing resistance are diverse, including viruses, bacteria, yeasts and fungi; some are plant pathogens and others not. The use of fungi to induce resistance in nonhost plants is well documented (Punja & Utkhede, 2003). Different strains of Fusarium oxysporum have been assayed as resistance-inducing agents. Some of these strains are nonpathogenic (Fuchs et al., 1997; Duijff et al., 1998; Larkin & Fravel, 1999; He et al., 2002; Elmer, 2004), but in other cases they are formae speciales able to infect hosts such as watermelon or tomato (Biles & Martyn, 1989; Huertas-González et al., 1999). In some cases there is evidence that F. oxysporum not only induced resistance, but also functioned as a competitor of other pathogenic Fusarium spp., thus conferring an additional protection mechanism (Fravel et al., 2003). Indeed, in most cases, nonpathogenic F. oxysporum has been tested for its ability to protect against pathogenic Fusarium spp., but few studies exist on the protection conferred against other pathogens, e.g. aerial pathogens, and systemic induction. Moreover, the physiology of the resistance induced by F. oxysporum is not clearly understood and should be accurately described before the system can be compared with other plant-pathogen models where the cascade of biochemical events is better known (Fravel et al., 2003). For these reasons, the ability of a strain of F. oxysporum f.sp. lycopersici, pathogenic to tomato, to confer protection in pepper (a nonhost) against three mycelial pathogens was tested. The interaction of F. oxysporum f.sp. lycopersici with a nonhost can serve as a tool to understand the mechanisms of this interaction. Thus, the physiological response of pepper was studied at the biochemical level by determining the activities of some of the enzymes involved in plant resistance and the levels of phenolic compounds, and by using an inhibitor of ethylene perception, a signal involved in resistance responses. The involvement of induced systemic resistance mechanisms in the protection conferred in pepper plants by F. oxysporum inoculation is discussed.

Materials and methods Fungal isolates and inoculum production Fusarium oxysporum f.sp. lycopersici (FOL) isolate CECT 2715 (ATCC 48112) was obtained from Colección Española de Cultivos Tipo (CECT). Verticillium dahliae isolate VDL was kindly provided by Dr Carlos Palazón (Servicio de Investigación Agroalimentaria, Zaragoza, Spain). Phytophthora capsici isolate UDC1PC was isolated in this laboratory in 1996 from a pepper plant showing phytophthora root rot symptoms. Botrytis cinerea strain B05·10 was kindly supplied by Dr Jan van Kan (Wageningen University, the Netherlands). Fusarium oxysporum f.sp. lycopersici conidia were obtained from liquid cultures in potato dextrose broth

grown at room temperature (20–25°C) for 10 days. Liquid cultures were filtered through gauze and the filtrate was centrifuged to precipitate the conidia. The conidia were resuspended in distilled water. This process of centrifugation and resuspension was repeated and the concentration of the final conidial suspension adjusted to 106 conidia mL−1. Verticillium dahliae conidia were obtained from cultures on potato dextrose agar (PDA) plates by gently flooding the dishes with sterile distilled water (Melouk, 1992). The concentration of the suspension was adjusted to 106 conidia mL−1. Phytophthora capsici was grown on V8-agar plates at 25°C in the dark for 7 days and then zoosporangia formation was induced in 10% (w/v) soil extract. After incubation for 45 min at 4°C and 30 min at room temperature, zoospores released from the zoosporangia were collected by filtering through gauze. The concentration of the inoculum was adjusted to 3000 zoospores mL−1. Botrytis cinerea strain B05·10 was cultured and inoculum prepared as described by Benito et al. (1998). Conidia were suspended at a concentration of 106 mL−1 in Gamborg’s B5 medium (Duchefa Biochemie BV, Haarlem, the Netherlands) supplemented with 10 mm glucose and 10 mm potassium phosphate (pH 6).

Plant material Pepper seeds were surface-disinfected by incubating in 10% (v/v) commercial bleach for 30 min. Seeds were then washed and soaked overnight in distilled water before being sown in sterile vermiculite. Unless otherwise stated, plants were grown at 25°C with a 16-h photoperiod. Two weeks after emergence, they were used in the experiments described below.

Preinoculation treatments Pepper plants were preinoculated with FOL by immersing the roots in a conidial suspension for 3 h. A control used sterile distilled water instead of inoculum. After preinoculation, pepper plants were transferred to small pots containing a mixture of vermiculite and potting soil (1:2, v/v). Where B. cinerea was used as the challenge pathogen, plants were subjected to additional treatments just before preinoculation. Some plants were exposed to 1methylcyclopropene (MCP), an inhibitor of ethylene perception, in a sealed container (Díaz et al., 2002) at a final concentration of 0·2 µL L−1. A control group of plants was kept in a container with no chemical added. Containers were opened after 4 h of treatment, and following aeration plants were then preinoculated as described above. Pepper plants (nine or 10 per treatment combination) were challenged 48 h after FOL preinoculation by inoculation with fungal pathogens of pepper. Alternatively, samples of 0·5–1 g were taken 48 h after preinoculation and stored at −20°C for analysis of enzyme activity or phenolic content. © 2005 BSPP Plant Pathology (2005) 54, 773–780

Fusarium protection against pepper pathogens

775

Challenge inoculation with Verticillium dahliae and disease evaluation

Effect of Fusarium on mycelial growth of pepper pathogens in vitro

Verticillium dahliae challenge inoculation was performed by cutting 1 cm off the apex of the roots and dipping the roots in the inoculum for 45 min. A control group was inoculated with water. Following inoculation, the plants were transferred to pots containing a mixture of vermiculite and potting soil (1:2, v/v). Experiments were performed twice. Disease incidence was determined periodically as the percentage of plants showing verticillium wilt symptoms. A symptom severity index was scored on the following scale: 0 (no symptoms), 1 (> 0 and ≤ 25% wilted leaves), 2 (> 25 and ≤ 50% wilted leaves), 3 (> 50 and ≤ 75% wilted leaves) and 4 (> 75% wilted leaves). In addition, the fresh weight and dry weight of the plants and the length of the epicotyl were measured 30 days after inoculation.

Dual cultures were used according to the method of Etebarian et al. (2000), with some modifications. All Fusarium–pepper pathogen combinations were assayed on PDA in 9 cm Petri dishes, with three to four replicate plates per treatment. Mycelial plugs, taken from actively growing colonies of FOL and one of the pepper pathogens tested, were placed 5 cm apart on the PDA. Plugs of V. dahliae were placed 5 days prior to those of FOL because of their quite different growth rates. Control plates consisted of two plugs of the same pathogen placed 5 cm apart on the PDA. Cultures were assessed daily, 1–2 days after pairing in the case of P. capsici and B. cinerea, and 1–5 days after pairing in the case of V. dahliae. Experiments were performed twice.

Challenge inoculation with Phytophthora capsici and disease evaluation For challenge inoculation with P. capsici, 5 mL of a zoospore suspension were poured uniformly over the surface of the soil in each pot. Experiments were performed three times. Disease incidence was determined 15 days after inoculation as the percentage of plants showing disease symptoms. Symptom severity was rated periodically on a 0–5 scale. AUDPC (area under the disease progress curve) values were calculated from the severity values according to the following formula (Campbell & Madden, 1990): n −1

AUDPC =

 yi + yi+1   (t i+1 − t i )  2

∑  i

where yi is the severity value for observation number i, ti is the number of days after inoculation at the moment of observation number i, and n is the number of observations.

Challenge inoculation with Botrytis cinerea and disease evaluation Botrytis inoculation was carried out as in Díaz et al. (2002). Botrytis cinerea conidia were suspended at a concentration of 106 mL−1 in Gamborg’s B5 medium (Duchefa Biochemie BV) supplemented with 10 mm glucose and 10 mm potassium phosphate (pH 6). The suspension was preincubated without shaking for 2–3 h. A 3 µL droplet of the suspension was placed on each leaf of the plant. Experiments were performed three times. Leaf infection by B. cinerea leads initially to the formation of primary necrotic lesions, but only some of these will expand, indicating fungal colonization of the leaf. The percentage of expanding lesions per plant and the area (mm2) of these were determined 72 h after inoculation. © 2005 BSPP Plant Pathology (2005) 54, 773–780

Test for colonization of pepper plants by Fusarium Plants were surface-disinfected by incubating for 15 min in 5 g sodium hypochlorite L−1, then washed with sterile distilled water and dried on sterile filter paper in a laminar flow cabinet. The different parts of the plant (roots, stem and leaves) were separated and placed on PDA plates. After 5–10 days of incubation at 25°C, the presence or absence of Fusarium colonies on each plate was recorded. Experiments were performed twice.

Extraction and determination of enzyme activities For β-1,3-glucanase extraction, samples were homogenized at 4°C in 0·5 m sodium acetate buffer (pH 5·2), containing 15 mm 2-mercaptoethanol. Crude extracts were centrifuged at 15 000 g and 4°C for 60 min. The supernatant was mixed with chilled acetone (1:4), kept at −20°C overnight, then centrifuged at 15 000 g and 4°C for 15 min. The pellet was washed twice with chilled acetone, dried and resuspended in 30 mm sodium acetate buffer (pH 5·2). The suspension was centrifuged again at 15 000 g and 4°C for 15 min and the supernatant used for β-1,3-glucanase determination according to the method of Kauffman et al. (1987). For chitinase and peroxidase extractions, samples were homogenized at 4°C in 0·1 m potassium phosphate buffer (pH 6·0) with the addition of 0·05 g polyvinylpolypyrrolidone (PVPP) per g fresh weight. Crude extracts were centrifuged at 10 000 g and 4°C for 20 min. Supernatants were desalted in a PD-10 column (Amersham Pharmacia Biotech, Spain) and the eluate analysed for enzyme activity. Chitinase activity was determined using the method of Wirth & Wolf (1990). Peroxidase activity was determined according to Ferrer et al. (1990). All experiments were performed twice.

Extraction and determination of phenolics All experiments were performed twice. Soluble phenolics were extracted with 80% methanol as described by Díaz

776

J. Díaz et al.

et al. (1997). The pellet obtained after the extraction of soluble phenolics was subjected to hydrolysis in 4 mL 4 n NaOH for 4 h at 37°C. After adjusting the pH to 1·0–2·0 with HCl, cell-wall-bound phenolics were extracted with diethyl ether. The organic phase was then evaporated to dryness and the residue dissolved in methanol. The phenolic content of each sample was determined using Folin-Ciocalteau reagent according to Díaz et al. (1997).

Statistical analyses All statistical analyses were performed using Statgraphics Plus for Windows, professional version 5·1 (Statistical Graphics Corp.). Where necessary, transformations were carried out to normalize the data prior to analysis. Data for percentage of diseased plants infected by P. capsici were analysed by logistic regression (P = 0·05) according to Agresti (1990). In the rest of the experiments where only two treatments were compared, a Duncan test was performed (P = 0·05). Data for percentage of expanding lesions caused by B. cinerea were analysed using the Kruskal–Wallis test (P = 0·05) followed by the multiple comparison procedure of Conover (1980). In the rest of the experiments with more than two treatments to be compared, a one-way anova was performed (P = 0·05), followed by Duncan tests for paired comparisons.

Results Challenge inoculation with Verticillium dahliae The percentage of inoculated plants showing symptoms increased to 100% 22 days after inoculation (Fig. 1a) compared with 60% in the FOL-preinoculated group. Severity index values increased with time in both plant groups, but whereas the control value increased up to 3·5 at 30 days after inoculation, the index for FOL-preinoculated plants did not increase further after reaching a value of 1·2 at 17 days after inoculation (Fig. 1b). Preinoculation with FOL, on its own, did not significantly change plant biomass, measured in terms of fresh weight, dry weight and stem length, relative to the water control (Table 1). Moreover, water content was not significantly affected by FOL. However, preinoculation with FOL significantly alleviated the reduction in biomass caused by Verticillium infection, the dry weight of these

Figure 1 Evolution of the incidence (a) and severity (b) of symptoms of disease caused by Verticillium dahliae in pepper (Capsicum annuum) plants preinoculated with Fusarium oxysporum f.sp. lycopersici (open circles) and control plants (filled circles). Data are means of two experiments. Vertical bars represent standard errors (P > 0.05).

plants not being significantly different from that of the uninoculated control (Table 1). The loss of water content caused by the challenge inoculation was also partially reversed by preinoculation with FOL.

Challenge inoculation with Phytophthora capsici There was a reduction of 37·7% in the percentage of plants showing symptoms of phytophthora root rot when preinoculated with FOL (Fig. 2a) and a reduction of nearly 50% in the AUDPC compared with the challenged control plants (Fig. 2b).

Table 1 Protection of pepper plants against Verticillium dahliae after preinoculation with Fusarium oxysporum f.sp. lycopersici (FOL) Preinoculation treatment

Challenge

Fresh weight (g)

Dry weight (g)

Stem height (mm)

Water content (%)a

n

H2O FOL H2O FOL

H2O H2O V. dahliae V. dahliae

2·216 a 2·004 a 0·558 c 1·368 b

0·149 a 0·141 a 0·047 b 0·120 a

72·6 a 71·6 a 42·7 c 55·3 b

100·0 a 90·6 a 24·3 c 59·5 b

19 20 20 20

Data are means of n plants from two experiments, 30 days after inoculation with V. dahliae. The total number of plants (n) analysed per treatment is indicated in the last column. Different letters within the same column indicate significant differences in a Duncan test (P ≤ 0·05) following square-root transformation of the data. a Water content expressed as a percentage of the double control pretreated and challenged with water.

© 2005 BSPP Plant Pathology (2005) 54, 773–780


Figure 2 Protection of pepper (Capsicum annuum) plants against Phytophthora capsici following preinoculation with Fusarium oxysporum f.sp. lycopersici (FOL). (a) Incidence (% of diseased plants) 15 days after inoculation; (b) severity of symptoms (AUDPC) 15 days after inoculation; H2O, plants pretreated with water and challenged with P. capsici; FOL, plants preinoculated with FOL and challenged with P. capsici. Data are means of three experiments. An asterisk indicates significant differences (P ≤ 0.05) relative to the water control on a logistic regression (percentage of diseased plants) or Duncan test (AUDPC).

Challenge inoculation with Botrytis cinerea In this case, not only was the effect of FOL preinoculation tested, but also the effect of pretreatment with an inhibitor of ethylene perception, MCP. FOL preinoculation significantly reduced the percentage of B. cinerea lesions undergoing expansion, but prior treatment with MCP reversed the effect of FOL. The percentage of expanding lesions for the treatment combination of MCP + FOL was not significantly different from the control, but it was reduced significantly by FOL alone (Table 2). Similarly, whereas preinoculation with FOL significantly reduced the area of lesions, MCP pretreatment reversed this effect.

Effect on in vitro growth of pathogens and on colonization of pepper plants by Fusarium Fusarium oxysporum f.sp. lycopersici did not significantly reduce the growth of any of the three pathogens in paired tests on agar plates (Fig. 3). In the cases of B. cinerea and V. dahliae, the diameter of the colony of the fungi paired with FOL was not significantly different from that of the

777

Figure 3 Effect of Fusarium oxysporum f.sp. lycopersici (FOL) on mycelial growth of pepper pathogens in vitro. Control, fungus paired with itself; FOL-paired, fungus paired with Fusarium oxysporum f.sp. lycopersici (FOL). Data for Phytophthora capsici and Botrytis cinerea were obtained 2 days after pairing, and for Verticillium dahliae 5 days after pairing. Data are means of two experiments. An asterisk indicates significant differences relative to the control in a Duncan test (P ≤ 0.05).

colonies of the same fungi paired together. The growth of P. capsici seemed to be slightly stimulated when paired with FOL after 2 days (Fig. 3). This stimulation of P. capsici growth was already apparent after 1 day of growth (data not shown). No FOL was re-isolated from plants inoculated with this fungus 48 or 144 h postinoculation.

Effect of Fusarium preinoculation on enzyme activities No differences in β-1,3-glucanase and peroxidase activities were observed between the water control and the FOLinoculated plants in the roots, stems or leaves 48 h after inoculation (Table 3). However, chitinase activity in the stems of FOL-inoculated plants was almost twice that of the control. In the case of roots and leaves, no clear differences in chitinase activity were observed between inoculated plants and the control.

Effect of Fusarium preinoculation on phenolics In the roots, no differences in soluble phenolics were observed between the control and the FOL-inoculated

Table 2 Protection of pepper plants against Botrytis cinerea after preinoculation with Fusarium oxysporum f.sp. lycopersici (FOL) Chemical pretreatment

Preinoculation treatment

Percentage expanding lesions per plant at 72 hpia

Total area of expanding lesions per plant at 72 hpi (mm2)b

n

Air Air MCP MCP

H2O FOL H2O FOL

50·0 a 37·5 b 58·3 a 59·5 a

135·3 a 70·5 b 184·0 a 183·2 a

30 30 30 29

Data are means of n plants from three experiments. The total number of plants (n) analysed per treatment is indicated in the last column. a hpi, hours postinoculation; different letters within the same column indicate significant differences between medians in a Kruskal–Wallis test (P ≤ 0·05) followed by the multiple comparison procedure of Conover (1980). b Different letters within the same column indicate significant differences in a Duncan test (P ≤ 0·05) following square-root transformation of the data.


778

J. Díaz et al.

Table 3 Effect of Fusarium oxysporum f.sp. lycopersici (FOL) inoculation of pepper on activities of enzymes related to plant defence in the different parts of the plant Roots

−1

−1

β-1,3-glucanase activity (mmol glucose s mg protein) Chitinase activity (mU mg−1 protein) Peroxidase activity (nkat mg−1 protein)

Stem

Leaves

H2O

FOL

H2O

FOL

H2O

FOL

44·7 ± 4·5 3·28 ± 0·45 57·6 ± 2·9

51·3 ± 0·1 3·08 ± 0·36 50·6 ± 1·1

12·9 ± 1·5 0·69 ± 0·16 25·7 ± 2·2

14·9 ± 1·7 1·21 ± 0·05 26·2 ± 2·9

18·3 ± 3·0 0·87 ± 0·05 5·5 ± 0·7

16·8 ± 4·5 1·04 ± 0·07 7·1 ± 1·0

Data are means ± SE from two experiments, 48 h after inoculation.

Table 4 Effect of Fusarium oxysporum f.sp. lycopersici (FOL) inoculation of pepper on phenolic content in the different parts of the plant Roots

Soluble phenolics (µg g−1 FW) Cell-wall-bound phenolics (µg g−1 FW)

Stem

Leaves

H2O

FOL

H2O

FOL

H2O

FOL

0·541 ± 0·004 0·177 ± 0·004

0·559 ± 0·032 0·203 ± 0·002

0·546 ± 0·004 0·141 ± 0·024

0·502 ± 0·016 0·219 ± 0·015

1·057 ± 0·045 0·283 ± 0·016

0·931 ± 0·067 0·320 ± 0·020

Data are means ± SE from two experiments, 48 h after inoculation.

plants 48 h after inoculation (Table 4). In stems and leaves, small reductions of 8 and 12%, respectively, were detected. There was an increase in cell-wall-bound phenolics in all organs of inoculated plants; the content of cell-wallbound phenolics in stems was 55% higher than in the water control, but values for roots and leaves were only 15 and 13% higher, respectively.

Discussion The protection induced by Fusarium against other Fusarium strains has been reported for several crops. The present work demonstrates that Fusarium is able to protect pepper plants from two other fungal and an oomycete pathogen. Although the protection was not complete, it was substantial in all cases, and it covered a broad range of pathogens. Indeed, systemic induction of resistance usually confers protection to a wide range of pathogens (KuÇ, 2000). The protection conferred by FOL could have been caused not only by induced resistance, but also by other effects, namely parasitism, antibiosis and competition. Thus far, there is no evidence of either parasitism or antibiosis among strains of F. oxysporum (Fravel et al., 2003). In the present work, parasitism was not studied, but antibiosis was not observed when FOL was paired with the other fungi in vitro. These results also make it unlikely that saprophytic competition for nutrients was involved, although some competition for the infection sites at the root surface was possible. It has been postulated that the root surface has a finite number of infection sites that could be protected if the inoculum density of Fusarium is high enough (Fravel et al., 2003). Such an effect is usually tested using the split-root method, where roots are divided into two parts, one in contact with the inducer and the other with the pathogen (Fuchs et al.,

1997; Larkin & Fravel, 1999). In the present experiments it was not possible to apply such a method because the roots were too small at the time of inoculation. Competition also seems to occur inside the plant, because nonpathogenic strains of Fusarium are able to actively colonize the surface of roots, penetrating the epidermal cells and colonizing the outer layer of cortical cells (Fravel et al., 2003). In the present experiments, however, it was not possible to isolate FOL from any of the parts of the plant at different times after inoculation, although the prolonged surface-sterilization procedure may have eliminated the fungus from superficial cells. In the case of Botrytis it is clear that neither competition nor antibiosis was possible because it is an aerial pathogen. Koike et al. (2001) observed the absence of antagonism in the protection induced by a nonpathogenic Fusarium isolate in cucumber against the aerial fungal pathogen Colletotrichum orbiculare. Biles & Martyn (1989) reported that inoculation with F. oxysporum f.sp. cucumerinum protected watermelon plants against infection by the aerial pathogen Colletotrichum lagenarium. Thus, the protection of FOL-induced pepper plants against Botrytis cannot be attributed to direct antagonism (competition, antibiosis), but must result from an induced resistance response. In the case of Verticillium and Phytophthora, both were exposed in the present experiments to the possibility of direct interaction with Fusarium, so antagonism by competition cannot be completely excluded, although in vitro assays did not show any direct antagonistic interaction. Duijff et al. (1998) suggested that the resistance induced by the nonpathogenic strain of F. oxysporum Fo47 may act through a classical SAR-like mechanism, because it induced PR proteins. In the present results, the protection conferred by FOL to B. cinerea was completely prevented by MCP, an inhibitor of ethylene perception. This suggests that the resistance induced by FOL was not © 2005 BSPP Plant Pathology (2005) 54, 773–780


through salicylate-dependent SAR, which is ethyleneindependent. On the other hand, the detection of chitinase in the stems of challenged plants in these experiments suggests that FOL did not induce a PGPR-ISR type response, since this is characterized by an absence of PR proteins (Pieterse & van Loon, 1999). The expression of PR proteins after induction with nonpathogenic Fusarium is well documented. Tamietti et al. (1993) found that tomato plants grown in soil steamed and amended with nonpathogenic Fusarium strains had higher chitinase activity than plants grown in steamed, unamended soil. Fuchs et al. (1997) reported increases in the activities of chitinase and β-1-3-glucanase in tomato plants inoculated with nonpathogenic F. oxysporum Fo47. Duijff et al. (1998) showed that this Fo47 strain induced the expression of chitinases and PR-1 at the protein level. Other authors using FOL (Recorbet et al., 1998) did not find any relationship between resistance and glucanases or chitinases, but did report a correlation of the enzymes with pathogenicity and disease. However, it must be noted that these results were obtained with tomato, a host susceptible to FOL, which is not the case for pepper. Peroxidase enzymes have also been implicated in induced resistance against pathogens (Hammerschmidt, 1999). Biles & Martyn (1993) and Martyn et al. (1996) found a correlation between the induction of resistance by F. oxysporum f.sp. niveum race 0 in watermelon and both increase in peroxidase activity and the appearance of a new acidic isozyme in the cotyledons. In the present case, peroxidases were not induced by Fusarium (Table 4) and the appearance of new isozymes was not observed (data not shown). He et al. (2002) found that nonpathogenic Fusarium oxysporum induced systemic resistance in Asparagus officinalis, but no increase in peroxidase was observed before challenge inoculation; plants induced with Fusarium and challenged with F. oxysporum f.sp. asparagi showed higher peroxidase activity than those not induced, then challenged. In pepper, challenge inoculation with P. capsici did not reveal such differences (data not shown). The present study showed increases in the levels of phenolic compounds after FOL inoculation. Tamietti et al. (1993) also found an increase in phenolics in tomato plants inoculated with nonpathogenic strains of Fusarium. Phenolics have been related to several functions involved in plant defence, namely preformed or inducible physical and chemical barriers against pathogens and local and systemic signalling for the expression of defence genes (Dixon et al., 2002). In the present work, salicylic acid, a signalling phenolic, was not measured specifically, so any comment on its role in the response of pepper to FOL would be speculative. However, it is remarkable that no changes in soluble phenolics were detected in pepper, in either the roots, stem or leaves. The levels of cell-wallbound phenolics were higher in FOL-treated plants, especially in stems, but this could be related to an increase in physical barriers against infection, as occurs with the response to elicitors in parsley (Conrath et al., 2001) and © 2005 BSPP Plant Pathology (2005) 54, 773–780

779

to pathogens in Arabidopsis (Tan et al., 2004), cases that have been more thoroughly studied. More detailed analyses are required to understand the exact nature of the phenolics induced by FOL in pepper and assign them physiological functions in plant defence. The results of this work suggest that induced systemic resistance requiring the ethylene signalling pathway and mediated by biochemical defence mechanisms such as chitinases and cell-wall-bound phenolics is involved in the nonhost response of pepper to FOL. More research is needed, however, to establish a complete picture of the physiological responses that lead to induced resistance.

Acknowledgements The authors are grateful to Dr Carlos Palazón (Servicio de Investigación Agroalimentaria, Zaragoza, Spain) for providing the VDL isolate and to Dr van Kan (Wageningen University, the Netherlands) for providing the B0510 isolate and MCP. This research was funded by the Xunta de Galicia (XUGA 10303A97 and PGDIT01AGR10301PR).

References Agresti A, 1990. Categorical Data Analysis. New York, USA: John Wiley. Benito EP, ten Have A, van’t Klooster JW, van Kan JAL, 1998. Fungal and plant gene expression during synchronized infection of tomato leaves by Botrytis cinerea. European Journal of Plant Pathology 104, 207–20. Bhat RG, Smith RF, Koike ST, Wu BM, Subbarao KV, 2003. Characterization of Verticillium dahliae isolates and wilt epidemics of pepper. Plant Disease 87, 789–97. Biles CL, Martyn RD, 1989. Local and systemic resistance induced in watermelons by formae-speciales of Fusarium oxysporum. Phytopathology 79, 856–60. Biles CL, Martyn RD, 1993. Peroxidase, polyphenoloxidase, and shikimate dehydrogenase isozymes in relation to tissuetype, maturity and pathogen induction of watermelon seedlings. Plant Physiology and Biochemistry 31, 499–506. Campbell CL, Madden LV, 1990. Introduction to Plant Disease Epidemiology. New York, USA: John Wiley. Conover WJ, 1980. Practical Nonparametric Statistics. New York, USA: John Wiley. Conrath U, Thulke O, Katz V, Schwindling S, Kohler A, 2001. Priming as a mechanism in induced systemic resistance of plants. European Journal of Plant Pathology 107, 113–9. Díaz J, Ros Barceló A, Merino de Cáceres F, 1997. Changes in shikimate dehydrogenase and the end products of the shikimate pathway, chlorogenic acid and lignins, during the early development of seedlings of Capsicum annuum. New Phytologist 136, 183–8. Díaz J, ten Have A, van Kan JAL, 2002. The role of ethylene and wound signaling in resistance of tomato to Botrytis cinerea. Plant Physiology 129, 1341–51. Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MSS, Wang LJ, 2002. The phenylpropanoid pathway and plant defence – a genomics perspective. Molecular Plant Pathology 3, 371–90. Duijff BJ, Pouhair D, Olivain C, Alabouvette C, Lemanceau P, 1998. Implication of systemic induced resistance in the suppression of fusarium wilt of tomato by Pseudomonas

780

J. Díaz et al.

fluorescens WCS417r and by nonpathogenic Fusarium oxysporum Fo47. European Journal of Plant Pathology 104, 903–10. Elmer WH, 2004. Combining nonpathogenic strains of Fusarium oxysporum with sodium chloride to suppress fusarium crown rot of asparagus in replanted fields. Plant Pathology 53, 751–8. Estrada B, Bernal MA, Díaz J, Pomar F, Merino F, 2000. Fruit development in Capsicum annuum: changes in capsaicin, lignin, free phenolics, and peroxidase patterns. Journal of Agricultural and Food Chemistry 48, 6234–9. Etebarian HR, Scott ES, Wicks TJ, 2000. Trichoderma harzianum T39 and T. virens DAR 74290 as potential biological control agents for Phytophthora erythroseptica. European Journal of Plant Pathology 106, 329–37. Ferrer MA, Calderón AA, Muñoz R, Ros Barceló A, 1990. 4-Methoxy-α-naphtol as a specific substrate for kinetic, zymographic and cytochemical studies on plant peroxidase activities. Phytochemical Analysis 1, 63–9. Fravel D, Olivain C, Alabouvette C, 2003. Fusarium oxysporum and its biocontrol. New Phytologist 157, 493–502. Fuchs JG, MoenneLoccoz Y, Defago G, 1997. Nonpathogenic Fusarium oxysporum strain Fo47 induces resistance to fusarium wilt in tomato. Plant Disease 81, 492–6. González-Salán MM, Bosland PW, 1991. Sources of resistance to verticillium wilt in Capsicum. Euphytica 59, 49–53. Hammerschmidt R, 1999. Induced disease resistance: how do induced plants stop pathogens? Physiological and Molecular Plant Pathology 55, 77–84. Hausbeck MK, Lamour KH, 2004. Phytophthora capsici on vegetable crops: research progress and management challenges. Plant Disease 88, 1292–303. He CY, Hsiang T, Wolyn DJ, 2002. Induction of systemic disease resistance and pathogen defence responses in Asparagus officinalis inoculated with nonpathogenic strains of Fusarium oxysporum. Plant Pathology 51, 225–30. Huertas-González MD, Ruiz-Roldán MC, Di Pietro A, Roncero MIG, 1999. Cross protection provides evidence for race-specific avirulence factors in Fusarium oxysporum. Physiological and Molecular Plant Pathology 54, 63–72. Kauffmann S, Legrand M, Geoffroy P, Fritig B, 1987. Biological function of pathogenesis-related proteins – 4 PR proteins of tobacco have 1,3-beta-glucanase activity. EMBO Journal 6, 3209–12. Koike N, Hyakumachi M, Kageyama K, Tsuyumu S, Doke N, 2001. Induction of systemic resistance in cucumber against several diseases by plant growth-promoting fungi: lignification and superoxide generation. European Journal of Plant Pathology 107, 523–33.

KuÇ J, 2000. Development and future direction of induced systemic resistance in plants. Crop Protection 19, 859–61. Larkin RP, Fravel DR, 1999. Mechanisms of action and dose–response relationships governing biological control of fusarium wilt of tomato by nonpathogenic Fusarium spp. Phytopathology 89, 1152–61. Martyn RD, Díaz Varela J, Kim D-H, 1996. Fusarium oxysporum f.sp. niveum race 2 transformed with race 0-DNA induces resistance and peroxidase activity in watermelon similar to wild type race 0. Phytopathology 86, S45. Melouk HA, 1992. Verticillium. In: Singleton, LL, Mihail, JD, Rush, CM, eds. Methods for Research on Soilborne Phytopathogenic Fungi. St Paul, MN, USA: APS Press, 175–8. Mercier J, Baka M, Reddy B, Corcuff R, Arul J, 2001. Shortwave ultraviolet irradiation for control of decay caused by Botrytis cinerea in bell pepper: induced resistance and germicidal effects. Journal of the American Society for Horticultural Science 126, 128–33. Oelke LM, Bosland PW, Steiner R, 2003. Differentiation of race specific resistance to phytophthora root rot and foliar blight in Capsicum annuum. Journal of the American Society for Horticultural Science 128, 213–8. Palloix A, Pochard E, Phaly T, Daubeze AM, 1990. Recurrent selection for resistance to Verticillium dahliae in pepper. Euphytica 47, 79–90. Parra G, Ristaino JB, 2001. Resistance to mefenoxam and metalaxyl among field isolates of Phytophthora capsici causing phytophthora blight of bell pepper. Plant Disease 85, 1069–75. Pieterse CMJ, van Loon LC, 1999. Salicylic acid-independent plant defence pathways. Trends in Plant Science 4, 52–8. Punja ZK, Utkhede RS, 2003. Using fungi and yeasts to manage vegetable crop diseases. Trends in Biotechnology 21, 400–7. Recorbet G, Bestel-Corre G, Dumas-Gaudot E, Gianinazzi S, Alabouvette C, 1998. Differential accumulation of β-1,3glucanase and chitinase isoforms in tomato roots in response to colonization by either pathogenic or non-pathogenic strains of Fusarium oxysporum. Microbiological Research 153, 257–63. Tamietti G, Ferraris L, Matta A, Gentile IA, 1993. Physiologicalresponses of tomato plants grown in Fusarium suppressive soil. Journal of Phytopathology 138, 66–76. Tan J, Bednarek P, Liu J, Schneider B, Svatoß A, Hahlbrock K, 2004. Universally occurring phenylpropanoid and speciesspecific indolic metabolites in infected and uninfected Arabidopsis thaliana roots and leaves. Phytochemistry 65, 691–9. Wirth SJ, Wolf GA, 1990. Dye-labeled substrates for the assay and detection of chitinase and lysozyme activity. Journal of Microbiological Methods 12, 197–205.
