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On-line version ISSN 1678-4677
Fitopatol. bras. vol.30 no.4 Brasília July/Aug. 2005
Potential of non-pathogenic Fusarium oxysporum isolates for control of Fusarium wilt of tomato
Potencial de isolados de Fusarium oxysporum não patogênico no controle da murcha de Fusarium do tomateiro
Juliano C. da SilvaI; Wagner BettiolII
IUNESP, Departamento de Produção Vegetal, Cx. Postal 237, CEP 18603-970, Botucatu, SP
IIEmbrapa Meio Ambiente, Cx. Postal 69, CEP 13820-000, Jaguariúna, SP, Fax: (019) 38678740, e-mail: firstname.lastname@example.org, Bolsista do CNPq
This study was done to evaluate the efficiency of non-pathogenic Fusarium oxysporum isolates (141/3, 233, 233/1, 245, 245/1, 251, 251/2, 251/5, and 257) in controlling vascular wilt caused by F. oxysporum f. sp. lycopersici, race 2 (isolates C-21A, TO11, and TO245) in tomato (Lycopersicon esculentum) cv. Viradoro seedlings. In order to determine the effect of non-pathogenic F. oxysporum isolates in tomato plants, the root system of 30-day-old seedlings was immersed in conidial suspensions (106 ml-1) of each isolate and the seedlings were transplanted to a cultivation substrate. Thirty-five days after transplanting it was observed that the non-pathogenic F. oxysporum isolates were not pathogenic to the cv. Viradoro nor did they affect seedling development. The efficiency of the non-pathogenic F. oxysporum isolates in controlling Fusarium wilt was determined by immersing the tomato seedling roots in the conidial suspension (106 ml-1) of each isolate and then transplanting them into substrates previously infested with isolates of F. oxysporum f.sp. lycopersici, race 2 (105 conidia ml-1 of substrate). Evaluations were performed 35 days after transplanting, for severity in scale with 1=healthy plant to 6=dead plant or plant showing vessel browning and wilted leaves up to the leader shoot and seedling height. The non-pathogenic F. oxysporum isolates were efficient in reducing the severity of the disease and maintaining normal plant development. These results provide evidence of the antagonistic activity of non-pathogenic F. oxysporum isolates in controlling vascular wilt caused by F. oxysporum f. sp. lycopersici race 2 in tomato.
Additional keywords: biological control, Fusarium oxysporum f.sp. lycopersici race 2, nonpathogenic F. oxysporum.
O trabalho avaliou a eficiência dos isolados (141/3, 233, 233/1, 245, 245/1, 251, 251/2, 251/5 e 257) de Fusarium oxysporum não patogênico ao tomateiro (Lycopersicon esculentum), no controle da murcha vascular causada por Fusarium oxysporum f. sp. lycopersici, raça 2 em plântulas de tomateiro cv. Viradoro. Para verificar o efeito dos isolados de F. oxysporum não patogênicos, o sistema radicular de plântulas de tomateiro, com 30 dias de idade, foi imerso na suspensão de conídios (106 ml-1) e as mudas transplantadas para substrato de cultivo. Após 35 dias do transplante foi verificado que esses isolados não foram patogênicos às plantas de tomateiro, nem afetaram o desenvolvimento das mudas. A eficiência dos isolados de Fusarium oxysporum não patogênicos no controle da murcha foi determinada imergindo-se as raízes de mudas de tomateiro em suspensão de conídios (106 conídios ml-1) e transplantando-as em substratos previamente infestados com os isolados de F. oxysporum f.sp. lycopersici, raça 2 (105 conídios ml-1 de substrato). Transcorridos 35 dias do transplante, foram realizadas as avaliações da severidade na escala de 1=planta sadia a 6=planta morta ou com vasos coloridos e folhas murchas até o ponteiro e altura das mudas. Os isolados de F. oxysporum não patogênicos foram eficientes em reduzir a severidade da doença e em manter normal o seu desenvolvimento. Esses resultados evidenciam a atividade antagônica dos isolados de F. oxysporum não patogênico no controle da murcha vascular do tomateiro, causada por Fusarium oxysporum f. sp. lycopersici raça 2.
Palavras-chave adicionais: controle biológico, Fusarium oxysporum f. sp. lycopersici, F. oxysporum não patogênico.
The tomato (Lycopersicon esculentum Mill.) is one of the world's most cultivated vegetable crops, and Brazil is one of the major producers. Tomato plants are affected by several diseases, including Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder & Hansen, which can cause serious economic losses. Methods used to control vascular wilt are either not very efficient or are difficult to apply. The best way to control the disease is by selecting resistant varieties of tomatoes. Although commercial varieties of tomato resistant to F. oxysporum f. sp. lycopersici races 1 and 2 are available, both additional pathogenic strains, and other races of the pathogen have been reported in several countries. For this reason, alternative methods of controlling the disease have been studied, with emphasis on biological control. Soils naturally suppressive to Fusarium wilt (Garibaldi et al., 1990; Alabouvette, 1999) have been reported in different regions of the world. Although several antagonistic microorganisms have been evaluated to control Fusarium wilt, the most promising are non-pathogenic F. oxysporum isolates (Rouxel et al., 1979; Garibaldi et al., 1987; Minuto et al., 1995ab). Saprophytic species of Fusarium have been found to be effective in reducing F. oxysporum in cyclamen (Cyclamen persicum Mill.), gerbera (Gerbera jamesonii Hook.), basil (Ocimum basilicum L.), asparagus (Asparagus officinalis L.), eggplant (Solanum melongena L.), carnation (Dianthus canyophyllus L.), watermelon [Citrullus lanatus (Thumb.) Matsumi & Nakai], tomato, chick pea (Cicer arietinum L.) and cucumber (Cucumis sativus L.) (Mandeel & Baker, 1991; Postma & Rattink, 1992; Yamagushi et al., 1992; Hervás et al., 1995; Minuto et al., 1995ab; Larkin & Fravel, 1999; He et al., 2002; Reid et al., 2002).
The objective of this work was to evaluate the efficiency of non-pathogenic F. oxysporum isolates for biological control of tomato wilt caused by F. oxysporum f. sp. lycopersici race 2.
Tomato cv. Viradoro, resistant to race 1 but susceptible to race 2 of F. oxysporum f. sp. lycopersici, was used in all assays. The tomato seedlings were produced for transplanting on Multihort® planting substrate in a styrofoam tray (35 mm × 35 mm) in a greenhouse.
The F. oxysporum f. sp. lycopersici race 2 isolates were supplied by Dr. Sami J. Michereff, Universidade Federal Rural de Pernambuco (isolate C-21A) and by Dr. Rômulo Fujito Kobori, Sakata Seed Sudamérica (isolates TO11 and TO245). The non-pathogenic F. oxysporum isolates 141/3, 233, 233/1, 245, 245/1, 251, 251/2, 251/5, and 257, isolated from carnation plants grown in suppressive soils in Italy, were supplied by Dr. Angelo Garibaldi, from Università degli Studi di Torino, Italy. The antagonistic isolates were introduced into Brazil through Laboratório de Quarentena Costa Lima (Brazil's official quarantine facility), of Embrapa Meio Ambiente (MA Proceeding no. 21052.011767/99-04). The inocula of all isolates were produced in potato-dextrose broth in shake culture (150 rpm), for ten days, at 25±2 ºC. The medium in flasks was seeded with 5mm diameter discs of PDA culture of the respective Fusarium sp. isolates. The culture was filtered through a double layer of sterilized gauze.
The plant growth substrate consisted of a soil and cattle manure (3:1 v/v) mixture. The soil (Yellow Latosol) showed the following chemical composition: P=5 mg dm-3; K=1.5, Ca=7, H+AL=95, BS=9.5, CEC=104.5 mmolc dm3; and V=9%. Each kilogram of the substrate was enriched with 0.2 g potassium chloride, 0.5 g single superphosphate and 6g dolomitic lime. The substrate was infested with respective isolates of F. oxysporum f. sp. lycopersici at concentrations of 103, 104, 105 and 106 conidia ml-1 of substrate, ten days prior to transplanting the 30-day-old seedlings. The plants were raised in the greenhouse for 35 days at which time disease severity and plant height were evaluated. The severity rating was done using the scale proposed by Tokeshi & Galli (1966), modified as follows: 1=healthy plant; 2=plant with brown vessels in the first internode region, without other visible symptoms; 3=plant with brown vessels up to the height of the first leaf, with yellowing of at least one leaflet; 4=plant showing vessel browning up to half of the stem length, with yellowing of two or more leaves; 5=plant showing vessel browning nearly to the leader shoot, with most leaves wilted, except the leader shoot; 6=dead plant or plant showing vessel browning and wilted leaves up to the leader shoot.
Effect of Fnp isolates on tomato
The root systems of tomato seedlings were washed in tap water, then immersed in a conidial suspension (106 ml-1) of respective non-pathogenic F. oxysporum isolates 141/3, 233, 233/1, 245, 245/1, 251, 251/2, 251/5, and 257, for 5 min. The seedlings were then transplanted to 500 ml pots containing the substrate. In addition to the non-pathogenic F. oxysporum isolates, the assay included a non-inoculated control and a control treated with the autoclaved PD culture medium. The plants were grown in the greenhouse and evaluations for disease severity and plant height were performed 35 days after transplanting, as previously described.
Effect of Fnp isolates on the control of Fusarium wilt in tomato
The tomato seedling root system was immersed in a conidial suspension (106 ml-1) of non-pathogenic F. oxysporum isolates 141/3, 233, 233/1, 245, 245/1, 251, 251/2, 251/5, and 257, for 5 min, after which the seedlings were transplanted to a substrate previously infested with a F. oxysporum f. sp. lycopersici isolates C-21A, TO11 and TO245 (105 ml-1 of substrates). The plants were grown in the greenhouse and evaluations for disease severity and plant height were performed 35 days after transplanting, as previously described.
A completely randomized experimental design with ten replicates was adopted for all assay. For the statistical analysis the data were transformed to sqrt (x + 0.5) and compared by the Tukey test at 5% probability, using the SAS System Software Package, version 8.
Race 2 of F. oxysporum f. sp. lycopersici isolates C-21A, TO11 and TO245 were found to be pathogenic to the cultivar Viradoro at all inoculum concentrations tested (Table 1), causing a drastic reduction of plant height. The isolate TO245 was the most virulent, causing the maximum diseases severity in plants grown in substrate infested with 106 and 105 conidia ml-1 of substrate. These results agree with those of Andrade & Micherref (2000), who demonstrated that tomato plants of different cultivars, inoculated with 106 conidia ml-1 of isolates C-1, C-7, C-21A, and F-23 of F. oxysporum f. sp. lycopersici race 2, showed a 50% disease incidence. He et al. (2002) also showed that 106 CFU g-1 soil of F. oxysporum f. sp. asparagi caused the death of asparagus plants.
Tomato seedlings whose root systems were immersed in the conidial suspension of non-pathogenic F. oxysporum isolates 141/3, 233, 233/1, 245, 245/1, 251, 251/2, 251/5, and 257, did not show symptoms of vascular diseases and developed normally. The non-pathogenic F. oxysporum isolates were obtained from carnation rhizospheres (Garibaldi et al., 1985), so were not pathogenic to the tomato plants. This is important because the same non-pathogenic F. oxysporum isolates can be useful for other hosts, as demonstrated by Minuto et al. (1995ab) for cyclamen and basil and by Garibaldi et al. (1990) for melon (Cucumis melo L.) and radish (Raphanus sativus L.).
When the tomato seedling root systems were immersed in inocula of non-pathogenic F. oxysporum isolates and the plants were grown in substract previously infested with race 2 of F. oxysporum f. sp. lycopersici isolates C-21A, TO11 and TO245, all non-pathogenic F. oxysporum isolates were efficient in controlling the disease; plants showed lower disease severity and greater height (Table 2), with no significant degree of difference between the non-pathogenic F. oxysporum isolates. These results agree with Garibaldi et al. (1987), Postma & Rattink (1992), and Minuto et al. (1995ab), who reported that non-pathogenic Fusarium spp. isolates, introduced by root immersion before transplanting, were efficient in colonizing the rhizosphere and in controlling Fusarium wilt. There are reports of non-pathogenic F. oxysporum that show they act by competing for infection sites and for nutrients, and by induction of resistance (Mandeel & Baker, 1991; Alabouvette & Couteaudier, 1992; Larkin & Fravel, 1999; Benhamou et al., 2001). In order to control vascular wilt caused by F. oxysporum f. sp. lycopersici, with non-pathogenic F. oxysporum it is necessary to study the best method for applying the non-pathogenic F. oxysporum, i.e., by treating the root systems by deepening or by applying the non-pathogenic F. oxysporum in soil/substrate in which the tomato is grown.
To Prof. Dr. Angelo Garibaldi, of Università degli Studi di Torino, for donating the non-pathogenic Fusarium oxysporum isolates; to Prof. Dr. Sami Jorge Michereff, of Universidade Federal Rural de Pernambuco and to Dr. Rômulo Fujito Kobori, of Sakata Seed Sudamérica, for donating the strain 2 Fusarium oxysporum f. sp. lycopersici isolates; and to Embrapa Hortaliças, for supplying the cv. Viradoro tomato seeds.
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Aceito para publicação em 02/03/2005
Autor para correspondência: Wagner Bettiol