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Efficacy of formaldehyde in the control of stem rot disease of cowpea in Nigeria
Efficacy o f f o r m a l d e h y d e in the control of s t e m rot d i s e a s e o f c o w p e a in N i g e r i a C. A. C. OKONKWO*
Department of Botany, University of Nigeria, Nsukka, Nigeria ABSTRACT. Laboratory and greenhouse studies were carried out to determine the effects of various concentrations (0-0025%, 0"005%, 0-01%, 0.1%, and 1.0%) of formaldehyde solution on the development of the stem-rot fungus, Sclerotium rolfsii. Laboratory tests were carried out on potato dextrose agar (PDA) in which the fungus was cultured. Significant differences were found in the mycelial growth rate and the number ofsclerotia produced by the fungus at various formaldehyde concentrations. The two highest concentrations (0.1% and 1.0%) totally inhibited the growth of the fungus. There was no significant difference in the effect of the lower concentrations (0"0025%, 0"005% and 0"01%) on fungal growth; sclerotia were formed at these levels. Results of greenhouse tests were similar to those obtained from laboratory tests: there was no stem-rot disease symptom at the two highest concentrations offorrnaldehyde, and at the lower concentrations where symptoms were manifest, there was no difference in the pathogenicity of the fungus to cowpea seedlings.
K~YWORDS:Formaldehyde; cowpea, stem rot; Sclerotiumrolfsii; Nigeria; chemical control
Introduction Cowpeas (Vigna unguiculata (L.) Walp) are important in world agriculture with > 5 x 106ha of land devoted to their production annually (Fery, 1981). However, like most crops with a long history of cultivation, they are subject to substantial annual losses as a result of pests and diseases (IITA, 1986), prominent among which is the stem rot disease due to infection by Sclerotium rolfsii Sacc. The chemical control of stem rot disease in the field has become practical only in the past two decades with the development of pentachloronitrobenzene (PCNB; quintozene) (Allam, Sinclair and Schilling, 1969). M a n y other fungicides, such as chloropicrin, methyl bromide, calcium cyanamide, a m m o n i a and formaldehyde, have also been used to check the disease (Higgins, 1934; Gould, 1954; Henis and Chet, 1967). With the establishment of these chemicals as fungicides for controlling some plant diseases, various workers have reported different concentrations for control. Abeygunawardena and Wood (1957) reported the use of 2.0% formaldehyde solution for the inhibition of germination ofsclerotia in the soil; Higgins (1934) reported 0-5% for effective control in Dutch iris; Gould (1954) also recorded 0-5% for bulbous iris. From the above information it appears that different host plants show various responses to different concentrations of formaldehyde solution. It appears also that so far no work has been done to investigate the efficacy of this chemical for the control of S. rolfsii on cowpea. The purpose of *Present address: Crop Production Programme, Abubakar Tafawa BalewaUniversity,PMB 0248, Bauchi,Nigeria
this study, therefore, was to determine the optimum concentration of formaldehyde solution for the control of stem rot disease of cowpea in Nigeria.
Materials and methods Formaldehyde ( H C H O ) solution (40% w/v) used in this investigation was manufactured by M a y and Baker Limited, Dagenham, England. From this standard solution concentrations of 0.0025, 0.005, 0-01,0-1 and 1.0% were made up with distilled water. Seeds of cowpea and culture of the test fungus, Sclerotium rolfsii Sacc., were supplied by the Botanical Garden and the Department of Botany, University of Nigeria, Nsukka, respectively. The effect of various concentrations of formaldehyde on radial and total growth of the fungus was determined. The radial growth was determined on solid medium (potato dextrose agar) in 9 cm Petri dishes (20 ml/dish). The total growth was determined in liquid dextrose medium without agar in 250 ml Erlenmeyer flasks (50 ml/flask). The inoculum for growth measurements for each Petri dish or Erlenmeyer flask consisted of a 2 m m disc of agar cut from the outer margin of a 3-day-old culture of the test fungus. The inoculated Petri dish and flasks were kept at room temperature (28 + 3°C). Colony diameters on solid media were measured when growth on the lowest formaldehyde concentration was about to cover the agar surface (4 days). Each measurement was taken as the average of two diameters of each culture. The cultures were further incubated for the production of sclerotia. The cultures in the liquid media were harvested after 9
o261-2194/89/o4/o28o--o3$03.00
© 1989Butterworth& Co (Publishers) Ltd CROP PROTECTION Vol. 8 August 1989, 280-282
C. A. C. OKONKWO days by filtering with suction on a piece of cloth attached to a Buchner funnel and washing with distilled water. Mycelial harvests were placed on pre-weighed filter papers dried in an oven at 90°C for 24 h, cooled in a desiccator over calcium chloride and weighed. Dry weights were obtained for five replicate treatments. • The inoculum for pathogenicity studies consisted of sand-maize meal medium inoculated with the test fungus. The sand-maize meal m e d i u m consisted of 95 g river sand, 5 g maize meal and 30 ml water. This mixture was sterilized by autoclaving for 50 min. When cooled, the medium was inoculated with a 3-day-old culture of S. rolfsii. For this inoculation, agar discs (2 mm) were cut from the periphery of the fungal colony and 20 such agar discs were placed in each Erlenmeyer flask containing the sterilized sand-maize meal medium. The discs were mixed with the medium and incubated at room temperature for 4 days before being used as inoculum. Cowpea seedlings were used in the assessment of pathogenicity of the fungus. The seedlings were grown in glazed clay pots (13.5 cm) containing soil mixed with compost. Each pot was filled with the soil to within 2 cm of the rim. The seeds were hand-planted in the pots and watered twice daily, in the morning and in the evening, until the seeds germinated. Some of the seedlings were later removed from the pots to avoid overcrowding. Ten days after the seeds were sown, the seedlings were inoculated with the fungus. The inoculum was distributed on the surface of the soil between the seedlings and a 1 em layer of sterilized river sand was added to the surface of each pot, and moistened. Equal volumes of the different formaldehyde concentrations (50 ml) were added as soil drenches to the pots. As in the laboratory tests, there were five replicates of each formaldehyde concentration. Control (check) pots were inoculated with sterilized sand-maize meal medium without S. rolfsii. The effects of the test concentrations of formaldehyde solutions on radial and total growth of the fungus, as well as its pathogenicity on cowpea seedlings, were analysed statistically, using Student's t test, to separate the mean effects.
281
0.1 or 1.0% (Table 1). No sclerotia were produced at the two higher levels of concentration, in which no mycelial growth occurred. Significant differences ( P = 0 - 0 1 ) were also noted in the dry weight of mycelium harvested from the three lower and the two higher concentrations of the medium 7 days after inoculation. The marginal increase in the dry weight of mycelium obtained at 0-0025% did not differ significantly (P = 0.01) from that obtained at concentrations of 0.005 or 0.01% (Table 1). Table 2 shows the results obtained from inoculation ,of the test cowpea seedlings. Assessment of fungal pathogenicity on cowpea seedlings in the greenhouse 'gave results similar to those of laboratory tests. The efficacy of the formaldehyde solution in disease control increased with increased concentration (0-1 and 1"0%). There was little control at the three lower concentration levels and no control at all in the check. The ecological success of many pathogenic fungi, including S. rolfsii, can be attributed in part to their ability to develop extensive mycelium during the growing season and to form resistant sclerotia during unfavourable conditions (Aycock, 1966; Rush and Lyda, 1982). Any chemical factors which affect the rate of growth of S. rolfsii or its ability to produce sclerotia could influence its survival. The results of this study confirm the reported toxicity of formaldehyde solution to S. rolfsii (Higgins, 1934; Gould, 1954; A b e y g u n a w a r d e n a and Wood, 1957). However, the use of formaldehyde in commercial control programmes has not been widely applied, except for high-value crops. This has often been attributed to
TABLE 1. Effect o f v a r i o u s c o n c e n t r a t i o n s o f f o r m a l d e h y d e Solution o n g r o w t h a n d s u r v i v a l o f Sclerotium rolfsii
Formaldehyde concn, (%)
Mean diameter of fungal c o l o n y after 4 d a y s (cm) ~
Mean number o f sclerotia produced after 14 d a y s a
Mean dry weight of mycelium (g)a
0"0025 0-05 0-01 0-1 1.0
8'9 a 8.6 a 8.1 a 0.0b 0.0 b
86 a 80 a 79 a 0b 0 b
0-692 a 0.622 a 0.590 a 0b 0 b
Each value is the mean of five replications. Values in the same column followed by the same letter are not significantly different according to Student's t test
a
Results
and
discussion
The effects of the various concentrations of formaldehyde solution on the radial growth of S. rolfsii, as measured by mean colony diameter after 4 days, are shown in Table 1. Significant differences ( P = 0-01) in radial growth of the fungus were observed between the three lower concentrations (0-0025, 0-005 and 0.01%) on the one hand and the two higher concentrations (0-1 and 1.0%) on the other. There was no growth at the 0.1 and 1-0% concentrations. The numbers of sclerotia produced 14 days after inoculation were significantly greater (P = 0.01) on the 0-0025, 0-005 and 0.01% media than at either
(P =
o.oi)
TABLE 2. Effects o f v a r i o u s c o n c e n t r a t i o n s o f f o r m a l d e h y d e solution o n the p a t h o g e n i c i t y ofS. rolfsii to c o w p e a seedlings 8 d a y s a f t e r i n o c u l a t i o n Formaldehyde concn(%)
No. o f p l a n t s in i n o c u l a t e d soil
Check O,0025 0,005 0,01 0,1 1.0
25 25 20 20 20 20
Wilted plants" (%) 100 80 75 75 0 0
a b b b c c
a Values followed by the same letter are not significantly different according to Student's t test (P = 0-01)
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Formaldehyde control of stem rot of cowpea
high cost; however, these results suggest that, for the practical control o f the stem rot disease c o m p l e x on c o w p e a seedlings, the use o f a low c o n c e n t r a t i o n (0.1%) o f the chemical will m i n i m i z e cost, a n d c o m p a r e s f a v o u r a b l y with results o f h i g h e r concentrations r e c o r d e d b y earlier workers (Higgins, 1934; G o u l d , 1954; A b e y g u n a w a r d e n a a n d W o o d , 1957). T h e low c o n c e n t r a t i o n ' o f - f o r m a l d e h y d e (0-1%) r e q u i r e d to check the g r o w t h o f the fungus suggests t h a t the m y c e l i u m should be the t a r g e t o f f o r m a l d e h y d e t r e a t m e n t . This could h a v e a direct effect on the timing o f field applications: b y a p p l y i n g the chemical as a soil d r e n c h a b o u t 10 days after planting, m u c h o f the m y c e l i u m t h a t w o u l d e v e n t u a l l y form sclerotia would be destroyed.
Acknowledgement T h e a u t h o r is v e r y grateful to Professor J . N . C . M a d u e w e s i , D e p a r t m e n t :of B o t a n y , U n i v e r s i t y o f Nigeria, Nsukka, f o r ~ v e r y carefully r e a d i n g a n d correcting the manuscript.
CROP PROTECTION Vol. 8 August 1989
References ABEYGUYAWARDENA,D. V. W. ANDWOOD,R. K. S. (1957). Effect of certain fungicides on Sclerotium rolfsii in the soil. Phytopathology 47, 607~509. ALLAM, A. I., SINCLAm, I. B. AND SCHILLING, P. E. (1969). Laboratory and greenhouse evaluation of four systemic fungicides. Phytopathology 59, 1659-1662. AYCOCK, R. (1966). Stem rot and other diseases caused by Sclerotium rolfsii. North Carolina Agriculture Experimental Station Bulletin 174, 17-133. FERY, R. L. (1981). Cowpea production in the United States. HortScience 16, 474. GOULD, G. J. (1954). Soil treatment tests for the control of Sclerotium rolfsii in bulbous iris. Phytopathology 44, 489~1~90. [-IENIS, Y. AND CHET, I. (1967). Mode of action of ammonia on Sclerotium rolfsii. Phytopathology 57, 425-427. HIGGINS,B. B. (1934). Physiology of parasitism ofSclerotium rolfsii Sacc. Phytopathology 24, 417-418. IITA (1986). International Institute of Tropical Agriculture, Annual Report and Research Highlights, pp. 49-50. Ibadan, Nigeria: IITA. RusH, C. M. ANDLYDA, S. D. (1982). Effects of anhydrous ammonia on mycelium and sclerotia of Phgmatotrichum omnivorum. Phytopathology 72, 1085-1089. Received~ 12 April 1987 Revised 2 June 1988 Accepted 5 December 1988
Department of Botany, University of Nigeria, Nsukka, Nigeria ABSTRACT. Laboratory and greenhouse studies were carried out to determine the effects of various concentrations (0-0025%, 0"005%, 0-01%, 0.1%, and 1.0%) of formaldehyde solution on the development of the stem-rot fungus, Sclerotium rolfsii. Laboratory tests were carried out on potato dextrose agar (PDA) in which the fungus was cultured. Significant differences were found in the mycelial growth rate and the number ofsclerotia produced by the fungus at various formaldehyde concentrations. The two highest concentrations (0.1% and 1.0%) totally inhibited the growth of the fungus. There was no significant difference in the effect of the lower concentrations (0"0025%, 0"005% and 0"01%) on fungal growth; sclerotia were formed at these levels. Results of greenhouse tests were similar to those obtained from laboratory tests: there was no stem-rot disease symptom at the two highest concentrations offorrnaldehyde, and at the lower concentrations where symptoms were manifest, there was no difference in the pathogenicity of the fungus to cowpea seedlings.
K~YWORDS:Formaldehyde; cowpea, stem rot; Sclerotiumrolfsii; Nigeria; chemical control
Introduction Cowpeas (Vigna unguiculata (L.) Walp) are important in world agriculture with > 5 x 106ha of land devoted to their production annually (Fery, 1981). However, like most crops with a long history of cultivation, they are subject to substantial annual losses as a result of pests and diseases (IITA, 1986), prominent among which is the stem rot disease due to infection by Sclerotium rolfsii Sacc. The chemical control of stem rot disease in the field has become practical only in the past two decades with the development of pentachloronitrobenzene (PCNB; quintozene) (Allam, Sinclair and Schilling, 1969). M a n y other fungicides, such as chloropicrin, methyl bromide, calcium cyanamide, a m m o n i a and formaldehyde, have also been used to check the disease (Higgins, 1934; Gould, 1954; Henis and Chet, 1967). With the establishment of these chemicals as fungicides for controlling some plant diseases, various workers have reported different concentrations for control. Abeygunawardena and Wood (1957) reported the use of 2.0% formaldehyde solution for the inhibition of germination ofsclerotia in the soil; Higgins (1934) reported 0-5% for effective control in Dutch iris; Gould (1954) also recorded 0-5% for bulbous iris. From the above information it appears that different host plants show various responses to different concentrations of formaldehyde solution. It appears also that so far no work has been done to investigate the efficacy of this chemical for the control of S. rolfsii on cowpea. The purpose of *Present address: Crop Production Programme, Abubakar Tafawa BalewaUniversity,PMB 0248, Bauchi,Nigeria
this study, therefore, was to determine the optimum concentration of formaldehyde solution for the control of stem rot disease of cowpea in Nigeria.
Materials and methods Formaldehyde ( H C H O ) solution (40% w/v) used in this investigation was manufactured by M a y and Baker Limited, Dagenham, England. From this standard solution concentrations of 0.0025, 0.005, 0-01,0-1 and 1.0% were made up with distilled water. Seeds of cowpea and culture of the test fungus, Sclerotium rolfsii Sacc., were supplied by the Botanical Garden and the Department of Botany, University of Nigeria, Nsukka, respectively. The effect of various concentrations of formaldehyde on radial and total growth of the fungus was determined. The radial growth was determined on solid medium (potato dextrose agar) in 9 cm Petri dishes (20 ml/dish). The total growth was determined in liquid dextrose medium without agar in 250 ml Erlenmeyer flasks (50 ml/flask). The inoculum for growth measurements for each Petri dish or Erlenmeyer flask consisted of a 2 m m disc of agar cut from the outer margin of a 3-day-old culture of the test fungus. The inoculated Petri dish and flasks were kept at room temperature (28 + 3°C). Colony diameters on solid media were measured when growth on the lowest formaldehyde concentration was about to cover the agar surface (4 days). Each measurement was taken as the average of two diameters of each culture. The cultures were further incubated for the production of sclerotia. The cultures in the liquid media were harvested after 9
o261-2194/89/o4/o28o--o3$03.00
© 1989Butterworth& Co (Publishers) Ltd CROP PROTECTION Vol. 8 August 1989, 280-282
C. A. C. OKONKWO days by filtering with suction on a piece of cloth attached to a Buchner funnel and washing with distilled water. Mycelial harvests were placed on pre-weighed filter papers dried in an oven at 90°C for 24 h, cooled in a desiccator over calcium chloride and weighed. Dry weights were obtained for five replicate treatments. • The inoculum for pathogenicity studies consisted of sand-maize meal medium inoculated with the test fungus. The sand-maize meal m e d i u m consisted of 95 g river sand, 5 g maize meal and 30 ml water. This mixture was sterilized by autoclaving for 50 min. When cooled, the medium was inoculated with a 3-day-old culture of S. rolfsii. For this inoculation, agar discs (2 mm) were cut from the periphery of the fungal colony and 20 such agar discs were placed in each Erlenmeyer flask containing the sterilized sand-maize meal medium. The discs were mixed with the medium and incubated at room temperature for 4 days before being used as inoculum. Cowpea seedlings were used in the assessment of pathogenicity of the fungus. The seedlings were grown in glazed clay pots (13.5 cm) containing soil mixed with compost. Each pot was filled with the soil to within 2 cm of the rim. The seeds were hand-planted in the pots and watered twice daily, in the morning and in the evening, until the seeds germinated. Some of the seedlings were later removed from the pots to avoid overcrowding. Ten days after the seeds were sown, the seedlings were inoculated with the fungus. The inoculum was distributed on the surface of the soil between the seedlings and a 1 em layer of sterilized river sand was added to the surface of each pot, and moistened. Equal volumes of the different formaldehyde concentrations (50 ml) were added as soil drenches to the pots. As in the laboratory tests, there were five replicates of each formaldehyde concentration. Control (check) pots were inoculated with sterilized sand-maize meal medium without S. rolfsii. The effects of the test concentrations of formaldehyde solutions on radial and total growth of the fungus, as well as its pathogenicity on cowpea seedlings, were analysed statistically, using Student's t test, to separate the mean effects.
281
0.1 or 1.0% (Table 1). No sclerotia were produced at the two higher levels of concentration, in which no mycelial growth occurred. Significant differences ( P = 0 - 0 1 ) were also noted in the dry weight of mycelium harvested from the three lower and the two higher concentrations of the medium 7 days after inoculation. The marginal increase in the dry weight of mycelium obtained at 0-0025% did not differ significantly (P = 0.01) from that obtained at concentrations of 0.005 or 0.01% (Table 1). Table 2 shows the results obtained from inoculation ,of the test cowpea seedlings. Assessment of fungal pathogenicity on cowpea seedlings in the greenhouse 'gave results similar to those of laboratory tests. The efficacy of the formaldehyde solution in disease control increased with increased concentration (0-1 and 1"0%). There was little control at the three lower concentration levels and no control at all in the check. The ecological success of many pathogenic fungi, including S. rolfsii, can be attributed in part to their ability to develop extensive mycelium during the growing season and to form resistant sclerotia during unfavourable conditions (Aycock, 1966; Rush and Lyda, 1982). Any chemical factors which affect the rate of growth of S. rolfsii or its ability to produce sclerotia could influence its survival. The results of this study confirm the reported toxicity of formaldehyde solution to S. rolfsii (Higgins, 1934; Gould, 1954; A b e y g u n a w a r d e n a and Wood, 1957). However, the use of formaldehyde in commercial control programmes has not been widely applied, except for high-value crops. This has often been attributed to
TABLE 1. Effect o f v a r i o u s c o n c e n t r a t i o n s o f f o r m a l d e h y d e Solution o n g r o w t h a n d s u r v i v a l o f Sclerotium rolfsii
Formaldehyde concn, (%)
Mean diameter of fungal c o l o n y after 4 d a y s (cm) ~
Mean number o f sclerotia produced after 14 d a y s a
Mean dry weight of mycelium (g)a
0"0025 0-05 0-01 0-1 1.0
8'9 a 8.6 a 8.1 a 0.0b 0.0 b
86 a 80 a 79 a 0b 0 b
0-692 a 0.622 a 0.590 a 0b 0 b
Each value is the mean of five replications. Values in the same column followed by the same letter are not significantly different according to Student's t test
a
Results
and
discussion
The effects of the various concentrations of formaldehyde solution on the radial growth of S. rolfsii, as measured by mean colony diameter after 4 days, are shown in Table 1. Significant differences ( P = 0-01) in radial growth of the fungus were observed between the three lower concentrations (0-0025, 0-005 and 0.01%) on the one hand and the two higher concentrations (0-1 and 1.0%) on the other. There was no growth at the 0.1 and 1-0% concentrations. The numbers of sclerotia produced 14 days after inoculation were significantly greater (P = 0.01) on the 0-0025, 0-005 and 0.01% media than at either
(P =
o.oi)
TABLE 2. Effects o f v a r i o u s c o n c e n t r a t i o n s o f f o r m a l d e h y d e solution o n the p a t h o g e n i c i t y ofS. rolfsii to c o w p e a seedlings 8 d a y s a f t e r i n o c u l a t i o n Formaldehyde concn(%)
No. o f p l a n t s in i n o c u l a t e d soil
Check O,0025 0,005 0,01 0,1 1.0
25 25 20 20 20 20
Wilted plants" (%) 100 80 75 75 0 0
a b b b c c
a Values followed by the same letter are not significantly different according to Student's t test (P = 0-01)
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Formaldehyde control of stem rot of cowpea
high cost; however, these results suggest that, for the practical control o f the stem rot disease c o m p l e x on c o w p e a seedlings, the use o f a low c o n c e n t r a t i o n (0.1%) o f the chemical will m i n i m i z e cost, a n d c o m p a r e s f a v o u r a b l y with results o f h i g h e r concentrations r e c o r d e d b y earlier workers (Higgins, 1934; G o u l d , 1954; A b e y g u n a w a r d e n a a n d W o o d , 1957). T h e low c o n c e n t r a t i o n ' o f - f o r m a l d e h y d e (0-1%) r e q u i r e d to check the g r o w t h o f the fungus suggests t h a t the m y c e l i u m should be the t a r g e t o f f o r m a l d e h y d e t r e a t m e n t . This could h a v e a direct effect on the timing o f field applications: b y a p p l y i n g the chemical as a soil d r e n c h a b o u t 10 days after planting, m u c h o f the m y c e l i u m t h a t w o u l d e v e n t u a l l y form sclerotia would be destroyed.
Acknowledgement T h e a u t h o r is v e r y grateful to Professor J . N . C . M a d u e w e s i , D e p a r t m e n t :of B o t a n y , U n i v e r s i t y o f Nigeria, Nsukka, f o r ~ v e r y carefully r e a d i n g a n d correcting the manuscript.
CROP PROTECTION Vol. 8 August 1989
References ABEYGUYAWARDENA,D. V. W. ANDWOOD,R. K. S. (1957). Effect of certain fungicides on Sclerotium rolfsii in the soil. Phytopathology 47, 607~509. ALLAM, A. I., SINCLAm, I. B. AND SCHILLING, P. E. (1969). Laboratory and greenhouse evaluation of four systemic fungicides. Phytopathology 59, 1659-1662. AYCOCK, R. (1966). Stem rot and other diseases caused by Sclerotium rolfsii. North Carolina Agriculture Experimental Station Bulletin 174, 17-133. FERY, R. L. (1981). Cowpea production in the United States. HortScience 16, 474. GOULD, G. J. (1954). Soil treatment tests for the control of Sclerotium rolfsii in bulbous iris. Phytopathology 44, 489~1~90. [-IENIS, Y. AND CHET, I. (1967). Mode of action of ammonia on Sclerotium rolfsii. Phytopathology 57, 425-427. HIGGINS,B. B. (1934). Physiology of parasitism ofSclerotium rolfsii Sacc. Phytopathology 24, 417-418. IITA (1986). International Institute of Tropical Agriculture, Annual Report and Research Highlights, pp. 49-50. Ibadan, Nigeria: IITA. RusH, C. M. ANDLYDA, S. D. (1982). Effects of anhydrous ammonia on mycelium and sclerotia of Phgmatotrichum omnivorum. Phytopathology 72, 1085-1089. Received~ 12 April 1987 Revised 2 June 1988 Accepted 5 December 1988