Biological control of fusarium diseases by fluorescent Pseudomonas and non-pathogenic Fusarium

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Biological control of fusarium diseases by fluorescent Pseudomonas and non-pathogenic

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Fusarium Philippe Lemanceau* and Claude A l a b o u v e t t e INRA, Station de Recherches sur la Flore Pathogdne dans le Sol, BV 1540, 21034 Dijon Cedex, France

Abstract

In soil-less culture of vegetables and flowers in greenhouses, fusarium diseases may induce severe damage. Under these growing conditions, biological control could be achieved by application of selected strains of fluorescent Pseudomonas or non-pathogenic Fusarium oxysporum. Seventy-four strains of fluorescent Pseudomonas were tested for their ability to reduce the incidence of fusarium wilt of flax when applied either alone or in association with one preselected non-pathogenic strain of Fusarium oxysporum (Fo47). Four classes were established, based on the effect of bacteria on disease severity, on their own or in association with Fo47. Most of the strains did not modify the percentage of wilted plants. However 10.8% of them, although haviag no effect on their own, significantly improved the control attributable to Fo47. One of these bacterial strains (C7) was selected for further experiments. Two trials conducted under commercial-type conditions demonstrated the effectiveness of the association of the bacterial strain C7 with the non-pathogenic Fusarium strain Fo47 to control fusarium crown and root rot of tomato, even when each antagonistic micro-organism was not efficient by itself. The yields were not significantly different in the protected plots in comparison with the healthy control.

Keywords

Biological control; fusarium wilt; fusarium crown and root rot; Fusarium oxysporum; fluorescent Pseudomonas; soil-less culture

Introduction In western Europe, the u,;e of soil-less culture to grow vegetables and flowers in glasshouses is rapidly increasing. The first objective of this cuLltural practice was the eradication of soil-borne pathogens; subsequently, the increase in the yield and in the precocity of the crop explain the extension of soil-less culture. In fact, although some ,diseases attributable to pathogenic fungi such as Pyrenochaeta lycopersici and Phomopsis sclerotioides are eradicated in soil-less culture by the use of inert growing substrates (e g. rockwool, glass fibre), other soil-borne fungi such as Pythium spp. and Fusarium oxysporum are still responsible for severe damage (Couteaudier and Lemanceau, 1989). Tomato plants may suffer from two different fusarium diseases: the plant wilt caused by Fusarium oxysporum f. sp. lycopersici and the crown and root rot caused by Fusarium oxysporum f. sp. radicis l)~copersici. This latter disease, described first in California (Leary and Endo, 1971) then in Japan (Sato and Araki, 1974), spread all over the world (Benhamou, Charest and Jarvis, 1989). It is now the most serious pathological problem in soil-less culture of tomato in greenhouses. Initially, the substrates are free from any micro-organism and are therefore very conducive to any *To whom correspondence should be addressed

pathogen introduced by aerial dissemination (Couteaudier and Alabouvette, 1981; Alabouvette, Couteaudier and Louver, 1982). However, this high level of receptiveness to introduced micro-organisms offers a unique opportunity to develop biological control by artificial inoculation of the substrate with selected antagonistic micro-organisms. The value of biological control of fusarium crown and root rot of tomato grown in soil-less culture is also considerable because no efficient fungicides nor resistant varieties usable in practice are available. Studies of soil naturally suppressive to fusarium wilts showed that two types of antagonistic micro-organisms are involved in the mechanisms of suppression. Strains of non-pathogenic Fusarium oxysporum isolated from the suppressive soil from Ch~teaurenard were effective to control fusarium wilts, when introduced into a conducive soil previously disinfested (Rouxel, Alabouvette and Louvet, 1979). Similarly, strains of fluorescent Pseudomonas isolated from the suppressive soil from the Salinas Valley were able to induce suppressiveness in conducive soils (Kloepper et al., 1980; Scher and Baker, 1982). Since these early reports many attempts have been made to use these antagonists to control fusarium wilts (Yuen, Schroth and McCain, 1985; Alabouvette et al., 1987) and in France a strain of non-pathogenic Fusarium is in the process of being registered. From a theoretical point of view the mode of action of

0261-2194/91/04/0279-08 © 1991 Butterworth-Heinemann Ltd

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Biological control of fusarium diseases: P. Lemanceau and C. Alabouvette

fluorescent Pseudomonas and non-pathogenic Fusarium are not mutually exclusive and they could be used simultaneously to control fusarium diseases. In fact, Park, Paulitz and Baker (1988) have shown that the association of fluorescent Pseudomonas with non-pathogenic strains of Fusarium was more effective to control fusarium wilt of cucumber than the use of each antagonist alone. Following the same approach, Lemanceau (1988) and Alabouvette (1989) established that the association of A12, a strain of Pseudomonas used by Scher and Baker (1982), and Fo47, a strain of non-pathogenic Fusarium, isolated from the soil from Chfiteaurenard, gave a better control of fusarium wilt of tomato than the use of the non-pathogenic Fusarium alone. These results led to the hypothesis that strains of Pseudomonas could improve the effectiveness of nonpathogenic Fusarium to control fusarium diseases. It seemed interesting, therefore, to screen fluorescent Pseudomonas not only for their antagonistic activity towards pathogenic Fusarium but also for their ability to control fusarium diseases in association with a non-pathogenic strain of Fusarium. The aim of this paper is to present the results of this screening and to show the potential benefit of an association of fluorescent Pseudomonas and nonpathogenic Fusarium to control fusarium crown and root rot of tomato in soil-less culture. One of our main objectives was to base our screening on the biological activity of the Pseudomonas in vivo rather than in vitro, because there is no clear relationship between the antagonism in vitro and in vivo. As we had a large collection of bacterial strains it was not possible to use tomato for the screening: the use of this plant would have been too space and time consuming. Moreover, flax is a model plant often used to study microbial interactions between antagonists and pathogenic Fusarium. Some strains of Pseudomonas selected with the flax model were then tested with tomato under standardized conditions. Two experiments with tomato were conducted under different conditions to demonstrate that the association of fluorescent Pseudomonas with non-pathogenic Fusarium was consistently efficient.

Inoculum of Fusarium

Two different kinds of inoculum were used, depending on the experiments. For the screening experiments, the pathogenic and non-pathogenic Fusarium were introduced into the rockwool (Grodan, The Netherlands) as 'talcinoculum'. This inoculum was produced according to the method described by Locke and Colhoun (1974) and modified by Tello-Marquina, Alabouvette and Louvet (1980). Microconidia of Fusarium produced in shake culture were mixed with talc, dried and stored at 4°C. Before use, talc-inoculum was suspended in water and inoculum density determined by plating on nutritive agar. Rockwool was infested by addition of a given volume of inoculum suspension to reach 2 × 107 c.f.u, ml- 1 substrate for the non-pathogenic Fusarium and 5 × 10 z c.f.u, ml- 1 substrate for the pathogenic Fusarium. In the biological control trials conducted under commercial-type conditions, both pathogenic and nonpathogenic Fusarium were introduced into the growing substrate as a suspension ofmicroconidia freshly harvested from a 5-day-old shake culture in malt extract (10 g 1- 1). To remove nutrients, conidia were washed three times in sterile demineralized water before their introduction into the rockwool at the concentration of 2 × l0 Tc.f.u, ml- 1 for the non-pathogenic Fusarium and 5 × 102 c.f.u, ml- 1 for the pathogenic Fusarium in the trial with hortifibre (Elf, Wogegal, France), a lignocellulosic growing substrate, and 3 × 10 5 c.f.u, ml 1 in the trial with rockwool. Strains of fluorescent Pseudomonas

Strains of Pseudomonas were isolated from the rhizosphere of tomatoes grown in rockwool and in the suppressive soil from Chfiteaurenard. Other strains were isolated from the rhizosphere of flax grown in the suppressive soils from Ch~teaurenard and Noirmoutier (France). Seventy-one strains from this collection were used during this experimentation, plus two strains (A12 and N1 R) isolated from American soils and provided by Dr R. Baker (Colorado State University) and one strain (PF36) isolated from a Swiss soil and provided by Dr G. Defago (Institut ffir Phytomedizin, Zurich). All the isolates were preserved by cryoconservation at - 80°C. Inoculum of fluorescent Pseudomonas

Materials and methods Strains of Fusarium

Two strains of pathogenic Fusarium were used: (l) a strain of F. oxysporum f. sp. lini (Foln3) inducing wilt of flax was used during the screening of Pseudomonas for their ability to reduce fusarium wilt severity; (2) a strain of F. oxysporum f. sp. radicis lycopersici (Forll9) inducing crown and root rot of tomato was used in all other experiments. The strain of non-pathogenic Fusarium oxysporum (Fo47) used was isolated from the suppressive soil from Chfiteaurenard. It has been extensively studied and is the most effective strain to control fusarium wilts of different crops (Corman et al., 1986).

CROP PROTECTION Vol. 10 August 1991

To infest rockwool, fluorescent Pseudomonas were grown for 48 h on solid medium of King (King, Ward and Raney, 1954). The bacteria were washed out from the agar and suspended in the nutrient solution used to water the growing substrate. Inoculum density was adjusted to I × 10 8 c.f.u, ml-1 of growing substrate after determination of the bacterial concentration of the suspension using a calibration curve determined by turbidity.

Experimental procedures: screening of fluorescent Pseudomonas Ability to reduce fusarium wilt severity. The 74 strains of Pseudomonas were used in the first experiment involving

Biological control of fusarium diseases: P. Lemanceau and C. Alabouvette

flax and its specific pathogen F. o. f. sp lini. Each strain was introduced to the rockwool plug, (a) alone, (b) in association with the pathogenic Fusarium or (c) in association with both the pathogenic and the non-pathogenic Fusarium. Infestation with the micro-organisms was made simultaneously with the first irrigation of the rockwool plugs. Flax (cultivar Regina susceptible to fusarium wilt), was sown in rockwool plugs held in racks. There were 12 plugs per treatment and one plant per plug. The plants were watered every day with an appropriate nutrient solution. The temperature of the growth chamber was regulated at 20°C during the night (8 h) and at 25°C during the day (16h). The number of wilted plants was recorded at the end of an 8-week culture period. Ability to reduce fusarium crown and root rot severity. As experiments dealing with crown and root rot of tomato are space and time consuming, only eight bacterial strains, from the initial collection of the strains of fluorescent Pseudomonas, were tested. They came from different origins and, depending on the strains selected, they were effective or ineffective against fusarium wilt of flax. As previously, each strain was added to a rockwool block, (a) alone, (b) in the presence of the pathogenic Fusarium or (c) in the presence of the pathogenic and non-pathogenic Fusarium. Tomatoes (cv. Montfavet 63.5) were grown separately in blocks of rockwool under standardized climatic conditions: 15°C during the night (8h), 20°C during the day (16h). Addition of the micro-organisms was made just before sowing the tomato seeds. After 8 weeks of culture, the plants were removed from the rockwool and symptoms on crown and roots were recorded. A pathological index was calculated by rating the symptoms of each plant on a scale from 0 for the healthy plant to 3 for the dead plant and then expressed as a percentage of the highest mark (3). Ability to achieve biological control of crown and root rot of tomato in greenhouses. The',most effective strain (C7) of fluorescent Pseudomonas in the last screening was chosen to control fusarium crown and root rot of tomatoes grown in soil-less culture under commercial-type conditions. As previously, the effectiweness of the bacterial strain C7 was assessed when introduced alone or in association with the non-pathogenic Fusarium Fo47. The experimental treatments were as follows: non-infested and infested control; the pathogenic Fusarium (Forl) with the fluorescent Pseudomonas (C7); Forl in association with the nonpathogenic Fusarium (Fo47); Forl in association with both C7 and Fo47. Two similar experiments were undertaken, the main difference being in the growing substrate used - rockwool in one case and hortifibre in the other. The first trial was carried out during the late summer and autumn and the second during the spring and early summer. Tomatoes (cv. Duranto) were sown in rockwool plugs, transferred 2 weeks later into rockwool blLocks and 1 month later set on pads of rockwool or hortifibre. Both antagonistic and

281

pathogenic micro-organisms were introduced at the same time as the setting of the plants on the pads of growing substrates. Plants were irrigated with a nutrient solution, the composition of which was modified according to the state of the crop and to the analysis of the drainage solution, according to the usual practice in commercial greenhouses. The number of wilted plants was recorded every week. The fruits were harvested and the commercial yield determined. There were three plants per pad and four replications of two pads per treatment. The experimental design was a randomized block assay. Statistical analysis Data were subjected to proportion comparisons by

Fisher's exact test (Miller, 1981) for the screening of Pseudomonas for their ability to reduce the fusarium wilt severity, or subjected to analysis of variance followed by Newman and Keuls' test (Kendall and Stuart, 1979) for all the experiments dealing with the fusarium crown and root rot.

Results Diversity among fluorescent Pseudomonas for their ability to reduce the incidence of fusarium wilt of flax

On the basis of their behaviour, the 74 strains of Pseudomonas studied were arranged in four classes. Figure 1 shows the behaviour of one strain representative of all the strains belonging to a class. The arrangement of the bacterial strains was based on their effectiveness against fusarium wilt of flax when applied alone and when applied in association with the non-pathogenic Fusarium. O f the strains, 67.6%, including the strains A12 and N 1R, belong to class 1: they do not affect significantly the incidence of wilt of flax either in the absence or the presence of the non-pathogenic Fusarium. Class 2 comprises 12.2% of the strains: they induce a significant decrease in disease incidence when they are introduced alone, but they do not interfere significantly with the control of the wilt attributable to the nonpathogenic Fusarium. Class 3 includes 10.8% of the strains: they show no effect when inoculated alone, but they induce a dramatic reduction in disease incidence when associated with the nonpathogenic Fusarium. Finally, 8.1% of the strains, including strain PF36, belong to class 4: they induce a significant decrease of wilt incidence either in the absence or the presence of the non-pathogenic Fusarium Fo47.

Ability of fluorescent Pseudomonas to control fusarium crown and root rot of tomato w h e n associated with Fo47

The results are shown in Figure 2. All eight bacterial strains improved the protection due to Fo47. This improvement is mainly noticeable for strains CH12, C7, C9, N2 and N5.

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Biological control of fusarium diseases: P. Lemanceau and C. A l a b o u v e t t e Class I

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the pathological index reached a level as high as 94.3 (Figure 4). The bacterial strain C7, on its own, reduced the percentage of wilted plants (Figure 3). The best biological control was determined by the association of both antagonistic micro-organisms C7 and Fo47 and by the nonpathogenic Fusarium on its own (Figures 3 and 4). The yield harvested in the non-infested control was not significantly different from the yield in the protected experimental treatments Fo47 or C 7 + Fo47 (Figure 5). The bacterial treatment alone gave some increase in yield but its benefi-

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Some strains seem to increase the disease severity but this increase is significant only for strain CH4. The only strain able to reduce disease severity significantly, on its own, is strain C7, which was therefore selected for further experiments. Biological control of fusarium crown and root rot of tomato The first experiment was conducted in a greenhouse where the tomatoes were cultivated on hortifibre during late summer and autumn: the results are shown in Figures 3, 4 and 5. The first wilted plants were recorded 9 weeks after the transfer of the plants to the pads (Figure 3). The disease incidence increased rapidly in the infested control. At the end of the experiment 91.7 % of the plants were wilted and

CROP PROTECTION Vol. 10 August 1991

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(Figures 6 and 7). When inoculated on their own, the non-pathogenic Fusarium or the fluorescent Pseudomonas did not significantly reduce the disease incidence (Figure 7); only the association of both antagonistic micro-organisms was able to control the disease efficiently. Furthermore, the plants so protected gave a yield that was not significantly different from that with the non-infested control (Figure 8). Although the yield with bacteria was significantly higher than that of the infested control, it was also significantly lower than that of the non-infested control.

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Biological control of fusarium diseases: P. Lemanceau and C. Alabouvette

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Discussion Biological control of fusarium crown and root rot of tomato can be achieved by introduction of either a non-pathogenic strain of Fusarium oxysporum, or a strain of fluorescent Pseudomonas, or by the association of both into the growing substrate. However, each treatment did not show the same effectiveness in reducing disease incidence. Non-pathogenic strains ofF. oxysporum have been used to induce soil suppressiveness to fusarium wilts (Rouxel et al., 1979; Tramier, Pionnat and Tebibel, 1983; Schneider, 1984; Garibaldi, Brunatti and Allochio, 1985; Tamietti and Alabouvette, 1986, Paulitz, Park and Baker, 1987). Among the strains isolated from the suppressive soil from Chfiteaurenard, the strain Fo47 is one of the most effective (Corman et al., 1986) and has been used in different trials for many years (Alabouvette et al., 1987; Alabouvette, 1989). The results presented in this paper corroborate the effectiveness of this strain in controlling not only fusarium wilt of flax, but also the fusarium crown and root rot of tomato. However, the effectiveness of the strain Fo47 was not similar in the two successive experiments conducted in the greenhouse. In the trial carried out during the autumn, biological control was excellent in spite of a high disease incidence in the infested control. On the other hand, strain Fo47 did not reduce disease severity significantly, in spite of its low level in the infested control during the spring trial. The variability of efficiency of Fo47 can be related to the different inoculum density ratio of non-pathogenic versus pathogenic Fusarium: in the first experiment this ratio was

C R O P PROTECTION Vol. 10 August 1991

very high (4 x 104) whereas it was deliberately very low (6.7 x 10) in the second experiment. It has already been established that the efficacy of non-pathogenic Fusarium depends not only on the characteristics of the strains, but mainly on their inoculum density which must determine a high non-pathogen/pathogen ratio (Couteaudier, 1989). Disease incidence of fusarium wilt is better correlated to this ratio than to the absolute density of the pathogen (Wensley and McKeen, 1963; Alabouvette, Couteaudier and Louvet, 1984). Disease severity is related to both inoculum potential and crop receptivity (Baker, 1968). Indeed, the difference in crop receptivity between the spring and autumn trials can account for the difference in disease incidence between the two experiments. Lower temperatures prevailing in the autumn were more favourable to disease development than were the higher temperatures during the spring. Crown and root rot of tomato is known to be favoured by temperatures < 20°C (Couteaudier and Alabouvette, 1989; Jarvis, 1989). Furthermore, the growing substrates used in both experiments were different: rockwool is a mineral and inert growing substrate whereas hortifibre is made of wood fibres able to sustain a certain amount of microbial development. Microbial analyses made at the end of cropping in the infested control suggested steady survival of introduced micro-organisms in rockwool but a clear increase in the inoculum density of the pathogen in hortifibre. The use of an antagonistic micro-organism belonging to the same species as the pathogen and having similar ecological requirements is of great interest, especially in organic substrates, because the two populations develop at similar rates, ensuring that the required ratio is maintained. This study showed also the large diversity among a collection of fluorescent Pseudomonas strains in their ability to control fusarium wilt. Most of the strains did not modify disease incidence, and only some of them reduced significantly the percentage of wilted plants when applied alone against F. oxysporum f. sp. lini. These results are in agreement with previous studies showing that only a low percentage of fluorescent Pseudomonas strains have a beneficial effect on plants (Burr, Schroth and Suslow, 1978; Kloepper et al., 1988). Furthermore, the screening experiments conducted in vivo permitted the establishment of a class of Pseudomonas strains ineffective on their own but improving biological control when associated with the non-pathogenic strain Fo47. This type of beneficial interaction previously described (Park et al., 1988; Alabouvette, 1989) appears to be more frequent than expected, and involved fluorescent Pseudomonas strains that did not have any antagonistic effect by themselves. The discrimination of the bacterial strains tested in classes was done after statistical comparison of the proportion of wilted plants between the controls (Foln3 and Foln3 plus Fo47) and the corresponding experimental treatments (Foln3 plus Pseudomonas and Foln3 plus Fo47 plus Pseudomonas). The significance of this analysis varies with the proportion of the wilted plants in the control. As this proportion was small in the control inoculated with both non-pathogenic and pathogenic Fusarium, the frequency of fluorescent

Biological control of fusarium diseases: P. Lemanceau and C. Alabouvette

Pseudomonas which induce a significant improvement of the protection was quite low. On the other hand, as the proportion of wilted plants was high in the infested control, minor reductions in disease severity by the fluorescent Pseudomonas were significant. It appears that a particular bacterial strain may not exert the same effect against two different fusarium diseases. The strains CH4, CH12 and R11, which were able to reduce significantly fusarium wilt severity on their own, did not determine the same beneficial effect on the severity of fusarium crown and root rot of tomato. On the other hand, strain C7 was never able to reduce by itself the incidence of fusarium wilt of flax, but it induced a clear effect when applied against crown and root rot of tomato. However, during two commercial-type experiments, the control exerted by this bacterial strain alone was not efficient enough to harvest a yield that did not differ from that of the non-infested control. Howew~r, the association of both the bacterial strain C7 and the non-pathogenic Fusarium strain Fo47 always provided the highest level of control, even when each micro-organism introduced separately did n o t induce a significant reduction of the disease severity. Nevertheless, when the control provided by Fo47 alone was excellent, it was obviously impossible to record a significant improvement of the control due to C7. The inconsistency of experiments dealing with biological control is a general problem. Our results illustrate the fact that the beneficial effect of fluorescent Pseudomonas can be proved only when growing conditions are not satisfactory, because of the activity of deleterious or pathogenic microorganisms (Suslow and Schroth 1982; Schippers, Bakker and Bakker, 1987; Weller, 1988). Previous studies have reported the effectiveness of the bacterial strains A 12 and N 1R to control fusarium wilts on their own or in association with non-pathogenic Fusarium (Scher and Baker, 1982; Pau:[itz et al., 1987; Alabouvette, 1989). In the present experinaents they did not show any beneficial effect at the end of an 8-week trial. However, after only 5 weeks, the pro Lection ensured by A12 and N1R, in the presence or the absence of non-pathogenic Fusarium, was significant. Their lack of effectiveness after 8 weeks could be attributable to their poor survival in our experimental conditions. Many papers devoted to the study of the interactions between pathogenic Fusarium and either fluorescent Pseudomonas or non-pathogenic Fusarium have provided several hypotheses of mechanisms, involving mainly competition for nutrients. Until now, only one hypothesis has been proposed to explain the mechanism of interaction between fluorescent Pseud~monas and non-pathogenic Fusarium (Park et al., 1988) but it is not supported by any published experimental data. Nevertheless, the association of C7 and Fo47 appears to be a very promising way to control the crown and root rot of tomato in soil-less culture.

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C. Tjamos and C. Beckman) pp. 457-478, NATO ASI Series, Vol. H28, Springer Vertag, Berlin

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Received 6 February 1990 Revised 24 October 1990 Accepted 8 January 1991