BACILLUS SUBTILIS FOR THE CONTROL OF POWDERY MILDEW ON CUCUMBER AND ZUCCHINI SQUASH

III. FITOSSANIDADE

BACILLUS SUBTILIS FOR THE CONTROL OF POWDERY MILDEW ON CUCUMBER AND ZUCCHINI SQUASH(¹)

WAGNER BETTIOL(²), ANGELO GARIBALDI(³) and QUIRICO MIGHELI(³)

ABSTRACT

Application of concentrated metabolites of Bacillus subtilis - CMBS - (5,000 µg/mL) one and 24 h before or after inoculation of Sphaerotheca fuliginea (3 x 10⁴ conidia/mL) reduced the number of lesions on cucumber leaves by 90-99%. The average number of lesions on control plants was 16.7 per leaf. A wettable powder product formulated with cells (10%) and metabolites (10%) of B. subtilis (WPBS), and CMBS sprayed on cucumber plants (1,000 µg/mL and 10,000 µg/mL) twice a week totally controlled powdery mildew. In the control treatment, 18 days after the first spray, the percent leaf surface covered by lesions was 99.0 and 46.7%, on the cotyledonary and expanded leaves, respectively. In the control treatment, 30 days after the first spray, the percent leaf surface with lesions was 26.1%, while leaves sprayed with CMBS presented no lesions. The fresh weight per plant was 4.3 g in the control treatment; 12.2 g, and 10.2 g for plants sprayed with CMBS at the concentration of 1,000 and 10,000 µg/mL, respectively; and 9.7 g and 10.1 g for plants sprayed with WPBS 1,000 and 10,000 µg/mL, respectively. For zucchini squash, CMBS (5,000 µg/mL) sprayed every 2, 4, and 6 days showed reductions in lesioned leaf surface of 100.0, 98.3, and 94.7%, respectively.

Index terms: biological control, cucurbit, Sphaerotheca fuliginea, Cucumis sativus, Cucurbita pepo.

RESUMO

CONTROLE DO OÍDIO DO PEPINO E DA ABÓBORA COM BACILLUS SUBTILIS

Aplicações de metabólitos concentrados produzidos por Bacillus subtilis - CMBS -(5.000 µg/mL), uma e 24 horas antes ou depois de inoculação de Sphaerotheca fuliginea (3 x 10⁴ conídios/mL), reduziram o número de lesões nas folhas de pepino entre 90 e 99%. O número médio de lesões nas plantas pulverizadas com água foi de 16,7 lesões por folha. Um produto pó-molhável, formulado com células (10%) mais metabólitos (10%) de Bacillus subtilis (WPBS) e CMBS pulverizados em folhas de pepino (1.000 µg/mL e 10.000 µg/mL) duas vezes por semana controlaram totalmente o oídio das folhas, enquanto no tratamento controle, 18 dias após a primeira pulverização, a porcentagem de área foliar coberta com o patógeno foi de 99,0 e 46,7% nas folhas cotiledonares e folhas verdadeiras respectivamente. No tratamento controle, 30 dias após a primeira pulverização, a porcentagem de área foliar com lesões foi de 26,1%, enquanto as folhas pulverizadas com CMBS não apresentavam lesões. A massa de matéria fresca por planta foi de 4,3 g para as plantas pulverizadas com água; 12,2 g e 10,2 g para as pulverizadas com CMBS, nas concentrações de 1.000 e 10.000 µg/mL respectivamente, e 9,7 g e 10,1 g para as pulverizadas com WPBS nas concentrações de 1.000 e 10.000 µg/mL respectivamente. Para abóbora, o CMBS pulverizado a cada 2, 4 e 6 dias, na concentração de 5.000 µg/mL, reduziu a porcentagem de área foliar coberta com o patógeno em 100,0, 98,3 e 94,7% respectivamente.

Termos de indexação: controle biológico, cucurbitáceas, Sphaerotheca fuliginea, Cucumis sativus, Cucurbita pepo.

1. INTRODUCTION

Cucurbit powdery mildew, caused by Sphaerotheca fuliginea (Schlecht.) Pollacci, is an important cucurbit disease world-wide. It occurs on leaves, stems and fruits, but usually is more abundant on upper leaf surfaces. Control methods currently available to growers include the use of resistant cultivars (Boiteux et al., 1995), repeated applications of sulphur (Kimati et al., 1980), and fungicides (Kimati et al., 1986). The constant use of fungicides, however, can result in environmental contamination. Marco et al. (1994) described the suppression of powdery mildew on squash by applications of whitewash, clay and antitranspirant materials. Unfortunately, the protection level achieved with these materials was lower than with a fungicide (propiconazole).

Several authors (Garibaldi et al., 1994; Reuveni et al., 1995) verified that powdery mildew was significantly better controlled by a single spray of aqueous solutions containing various phosphates and potassium salts. These authors concluded that phosphates and potassium are appropriate leaf fertilizers, with a potential for disease control.

Biological control agents for cucurbit powdery mildew have been described. The most important ones are: Tilletiopsis spp. (Hijwegen, 1992; Urquhart et al., 1994), Ampelomyces quisqualis (Falk et al., 1995), Cladosporium sp. (Minuto et al., 1991), Verticillium lecanii (Verhaar & Hijwegen, 1993), and Acremonium alternatum (Malathrakis, 1985). As far as we know, no references are available on the biological control of S. fuliginea by Bacillus subtilis, which is effective in the control of other plant pathogenic fungi (Baker et al., 1983; Pusey & Wilson, 1984; Bettiol et al., 1992, 1994). This study aimed at evaluating the potential of concentrated metabolites (CMBS) and a product formulated with cells and metabolites (WPBS) of B. subtilis as to the control of powdery mildew on cucumber and zucchini squash.

2. MATERIAL AND METHODS

1. Experimental products used

Two experimental products with B. subtilis were tested for the control of S. fuliginea:

a) CMBS - concentrated metabolites of B. subtilis, isolate AP-3 (Bettiol & Kimati, 1990). B. subtilis was grown in Erlenmeyer flasks (1 L capacity) containing dextrose 1%, peptone 1%, yeast extract 0.5%, NaCl 0.3%, KH₂PO₄ 0.1% and MgSO₄ 0.05%, placed on a rotary shaker at 150 rpm and 27 ± 2^o C for 7 days. The fermentation medium was centrifuged at 13,200 g to remove bacteria cells. The antibiotics were precipitated from the supernatant by adjusting pH to 2.0 with HCl 1N. The resulting material was again centrifuged at 13,200 g, being the metabolites stored in bottles at 2^o C.

b) WPBS - a wettable powder of B. subtilis was formulated with cells (60 g) and concentrated metabolites (60 g) of B. subtilis, clay (480 g - Sintermor Mineração Ltd.), surfactant (7.92 g - Surfon 950 PM - Oxiteno S.A.), and water (2.4 L). This mixture was crushed, sieved (0.210 mm) and dried (Spray-dryer set at 101 ± 1^o C, at entry).

2. Effect of timing of CMBS application on severity of S. fuliginea attacks on cucumber

Cucumber plants (Marketmore Standard) were grown in polyethylene pots of 0.6 L containing a peat-based organic substrate until they reached the developmental stage of two expanded leaves. At this stage, plants were sprayed with CMBS (5,000 µg/mL) one and 24 h before or after inoculation with S. fuliginea (3 x 10⁴ conidia/mL). Control plants were sprayed with water and, after 1 h, inoculated with the pathogen. The application was performed with a De Vilbiss # 15 hand sprayer until runoff. Experiment was set in a randomized block design with five replications per treatment with a replication consisting of one pot, containing five plants each.

After inoculation the plants were placed in a growth chamber (25 ± 2^o C, 12 h light-6,000 lux). The plants were evaluated after 13 days by counting the number of lesions per leaf for the expanded leaves and by estimating the cotiledonary leaf surface covered by powdery mildew (Marco et al., 1994).

3. Effect of WPBS and CMBS concentrations on severity of S. fuliginea on cucumber

Cucumber plants (Mezzolungo-Marketer) seven days old, grown as described previously, were transferred to a growth chamber, with high S. fuliginea inoculum potential and sprayed twice a week with: a) water; b) CMBS (1,000 µg/mL); c) CMBS (10,000 µg/mL); d) WPBS (1,000 µg/mL); and e) WPBS (10,000 µg/mL). The plants were evaluated 18 and 30 days after the first spray by estimating the leaf surface covered by lesions. Fresh weight of the plants was evaluated at the end of the experiment. The experiment was set in a randomized block design, with five replications. Before the first disease evaluation, pots containing five plants had the number reduced to two plants for each pot.

4. Effect of spray frequency of CMBS on the effectiveness of S. fuliginea control on zucchini squash

Zucchini squash plants (IBIS NUOVO 423 F1), with three expanded leaves were grown in polyethylene pots of 2.0 L containing a peat based organic substrate, in a growth chamber (25 ± 2^o C, 12 h light) with high S. fuliginea inoculum potential. These plants were sprayed with CMBS 5,000 µg/mL every 2, 4 and 6 days. Control plants were sprayed every two days with distilled water. The experiment was set in a randomized block design with five replications and two plants per pot.

The leaf surface with lesions was evaluated 8, 13 and 20 days after the first spray, and the fresh weight of leaves was evaluated 21 days after the first spray.

3. RESULTS AND DISCUSSION

On cucumber cotyledonary leaves CMBS sprayed one and 24 h before or after inoculation of S. fuliginea provided 100% of disease control, while the percent leaf surface covered by lesions in the control was 5.7%. On fully expanded leaves the number of lesions on the control was 16.8 per leaf (Table 1), while the reduction of lesion number by CMBS sprayed 1 h before, 1 h after, and 24 h after inoculation were 99.4, 98.1, and 99.5%, respectively; CMBS sprayed 24 h before inoculation caused a reduction of 89.5%. Such an effect was possible due to the ectoparasitic habit of the pathogen similar results were obtained when Cylindrocladium scoparium, Cur-vularia eragrostidis and Hemileia vastatrix were inoculated before B. subtilis application, with no effective control observed (Bettiol et al., 1988; Bettiol & Varzea, 1992; Andrade et al., 1995). The result obtained with CMBS on S. fuliginea, however, indicates a broader range of timing for effective application with respect to the life cycle of the fungus.

CMBS and WPBS (1,000 and 10,000 µg/mL) sprayed on cucumber plants twice a week totally controlled the powdery mildew. In the control treatment the percent leaf surface covered by lesions 18 days after the first spray was 99.0 and 46.7% on the cotyledonary and expanded leaves, respectively (Table 2). Compared to the control plants, 30 days after the first spray the percent leaf surface with lesions and percent leaf surface with lesion per injuried leaf were 26.1 and 43.5%, respectively. The plants sprayed with CMBS showed no lesions. The fresh weight per plant was 4.3 g in the control plants, 12.2 g and 10.2 g for the plants sprayed with CMBS 1,000 and 10,000 µg/mL, respectively; 9.7 and 10.1 g for the plants sprayed with WPBS 1,000 and 10,000 µg/mL, respectively (Table 2). As no disease occurred after CMBS and WPBS application, no statistical analyses were performed on the disease variables of this experiment.

As far as plant fresh weight is concerned, treatments differed significantly (P < 0.05) from the control, and both CMBS and WPBS at 1,000 µg/mL and spray twice a week effectively increased plant weight.

When sprayed with CMBS every 2, 4 and 6 days zucchini squash showed reductions in leaf surface covered by lesions of 100.0, 98.3, and 95.5%, respectively. Considering the leaf surface with lesions per injuried leaf the values were 100.0, 96.9, and 91.9% for plants sprayed every 2, 4, and 6 days, respectively. The fresh weight for the control plot was 6.4 g/plant while for the plants sprayed every 2, 4, and 6 days were 13.5, 12.8, and 15.2 g/plant, respectively (Table 3).

The results obtained indicated the efficacy of both products, CMBS and WPBS of B. subtilis, in controlling S. fuliginea on cucumber and zucchini squash. Further studies are needed to characterize the mechanism of action of such products and the role of antibiotic metabolites produced by B. subtilis, which could be the major responsible for the biocontrol achieved.

The efficacy of CMBS and WPBS for controlling powdery mildew (S. fuliginea) on cucumber and zucchini squash has been found to be similar to those observed by Bettiol et al. (1992, 1994) for Uromyces appendiculatus var. appendiculatus and H. vastatrix, and by Lazzaretti & Bettiol(⁴) for controlling several pathogens associated with rice, wheat, bush-bean and soybean seeds. Such efficacy corroborates the recognized capability of B. subtilis' metabolites for inhibiting many phytopathogenic fungi (Baker et al., 1983; McKeen et al., 1986; Pusey, 1989; Thirumalachar & O'Brien, 1977). These results indicate that, given the high-performance of B. subtilis metabolites against the studied disease on zucchini squash and cucumber, the CMBS and WPBS metabolite-compounds may contribute for the control of other pathogens associated to these crops.

ACKNOWLEDGEMENTS

The authors express sincere gratitude to Dr. G.S. Rodrigues for critically reviewing the manuscript.

⁽²) Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Centro Nacional de Pesquisa de Monitoramento e Avaliação de Impacto Ambiental (CNPMA). Caixa Postal 69, 13820-000 Jaguariúna (SP).

⁽³) Università degli Studi di Torino, Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali-Patologia Vegetale, Via Leonardo da Vinci, 44, 10095 Grugliasco (TO), Italy.

• ANDRADE, D.E.G.T.; SILVA, E.B.; MICHEREFF, S.J.; MARIANO, R.L.R. & BETTIOL, W. Controle da queima das folhas de inhame com extratos e células formuladas de Bacillus subtilis. Summa Phytopathologica, Jaguariúna, 21:202-205, 1995.
• BAKER, C.J.; STAVELY, J.R.; THOMAS, C.A.; SASSER, M. & MACFALL, J.S. Inhibitory effect of Bacillus subtilis on Uromyces phaseoli and on development of rust pustules on bean leaves Phaseolus vulgaris. Phytopathology, St. Paul, 73:1148-1152, 1983.
• BETTIOL, W.; AUER, C.G.; CAMARGO, L.E.A. & KIMATI, H. Controle da mancha foliar de Eucalyptus grandis e E. urophylla induzida por Cylindrocladium scoparium com Bacillus subtilis. Summa Phytopathologica, Jaguariúna, 14:210-218, 1988.
• BETTIOL, W.; BRANDĂO, M.S.B. & SAITO, M.L. Controle da ferrugem do feijoeiro com extratos e células formu-ladas de Bacillus subtilis. Summa Phytopathologica, Jaguariúna, 18:153-159, 1992.
• BETTIOL, W. & KIMATI, H. Efeito de Bacillus subtilis sobre Pyricularia oryzae agente causal da brusone do arroz. Pesquisa agropecuária brasileira, Brasília, 25(8):1165-1174, 1990.
• BETTIOL, W.; SAITO, M.L. & BRANDĂO, M.S.B. Controle da ferrugem do cafeeiro com produtos ŕ base de Bacillus subtilis. Summa Phytopathologica, Jaguariúna, 20:119-122, 1994.
• BETTIOL. W. & VARZEA, V.M.P. Controle biológico da ferrugem (Hemileia vastatrix) do cafeeiro com Bacillus subtilis em condiçőes controladas. Fitopatologia Brasileira, Brasilia, 17:91-95, 1992.
• BOITEUX, L.S.; REIFSCHNEIDER, F.J.B. & PESSOA, H.B.S.V. Phenotypic expression of quantitative and qualitative components of partial resistance to powdery mildew (Sphaerotheca fuliginea race 1) in melon (Cucumis melo) germplasm. Plant Breeding, Berlin, 114:185-187, 1995.
• FALK, S.P.; GADOURY, D.M.; PEARSON, R.C. & SCEM, R.C. Partial control of grape powdery mildew by the mycoparasite Ampelomyces quisqualis. Plant Disease, St. Paul, 79:483-490, 1995.
• GARIBALDI, A.; ALOI, C. & MINUTO, A. Osservazioni sull'attivitŕ di prodotti fosfatici nei riguardi di Erysiphe sp. su pomodoro in coltura protetta. Atti Giornate Fitopatologiche, Bologna, 3:245-250, 1994.
• HIJWEGEN, T. Biological control of cucumber powdery mildew with Tilletiopsis minor under greenhouse conditions. Netherlands Journal of Plant Pathology, Wageningen, 98:221-225, 1992.
• KIMATI, H.; CARDOSO, C.O.N. & BERGAMIN FILHO, A. Doenças das cucurbitáceas (abóbora, abobrinha, chuchu, melancia, melăo, moranga, pepino). In: Galli, F., ed. Manual de Fitopatologia: doenças das plantas cultivadas. Săo Paulo, Ceres, 1980. p. 251-269.
• KIMATI, H.; SOAVE, J.; ESKES, A.B.; KUROZAWA, C.; BRIGNANI NETO, F. & FERNANDES, N.G. Guia de fungicidas agrícolas Piracicaba, Livroceres, 1986. 281p.
• McKEEN, C.D.; REILLY, C.C. & PUSEY, P.L. Production and partial characterization of antifungal substances antagonistic to Monilinia fructicola from Bacillus subtilis. Phytopathology, St. Paul, 76:136-139, 1986.
• MALATHRAKIS, N.E. The fungus Acremonium alternatum Linc. Fr., a hyperparasite of the cucurbits powdery mildew pathogen Sphaerotheca fuliginea. Zeitschrift fur Pflanzenkankheiten und Pflanzenschutz, Stuttgart, 92:509-515, 1985.
• MARCO, S.; ZIU, O. & COHEN, R. Suppression of powdery mildew in squash by applications of whitewash, clay and antitranspirant materials. Phytoparasitica, Bet Dagan, 22:19-29, 1994.
• MINUTO, G.; GARIBALDI, A. & GULLINO, M.L. Antagonistic activity of some microorganisms against powdery mildew (Sphaerotheca fuliginea) of zucchini: preliminary results. Bulletin OILB/SROP, Alassio, 14:181-186, 1991.
• PUSEY, P.L. Use of Bacillus subtilis and related organisms as biofungicides. Pesticide Science, London, 27:133-140, 1989.
• PUSEY, P.L. & WILSON, C.L. Postharvest biological control of stone fruit brown rot by Bacillus subtilis. Plant Disease, St. Paul, 68:753-756, 1984.
• REUVENI, M.; AGAPOV, V. & REUVENI, R. Suppression of cucumber powdery mildew (Sphaerotheca fuliginea) by foliar sprays of phosphate and potassium salts. Plant Pathology, Cambridge, 44:31-39, 1995.
• THIRUMALACHAR, M.J. & O'BRIEN, M.J. Suppression of charcoal rot in potato with a bacterial antagonist. Plant Disease Reporter, St. Paul, 61:543-546, 1977.
• URQUHART, E.J.; MENZIES, J.G. & PUNJA, Z.K. Growth and biological control activity of Tilletiopsis species against powdery mildew (Sphaerotheca fuliginea) on greenhouse cucumber. Phytopathology, St. Paul, 84:341-351, 1994.
• VERHAAR, M.A. & HIJWEGEN, T. Efficient production of phyaloconidia of Verticillium lecanii for biocontrol of cucumber powdery mildew, Sphaerotheca fuliginea. Netherlands Journal of Plant Pathology, Wageningen, 99:101-103, 1993.

⁽¹

) Received for publication in December 2

^nd, 1996 and accepted in June 6, 1997.

⁽⁴

) LAZZARETTI, E. & BETTIOL, W. Tratamento de sementes de arroz, trigo, feijão e soja com produto formulado à base de células e de metabólitos de

Bacillus subtilis. Scientia Agricola, Piracicaba (in press).

Publication Dates

• Publication in this collection
  16 June 1999
• Date of issue
  1997

History

• Accepted
  06 June 1997
• Received
  02 Dec 1996