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Collection of plant material
Surveys were conducted in farmers’ fields in six villages in the Ouémé valley (Agbonou, Agonguey, Agonlin, Dannou, Gangban and Wozounme) during the 2001 and 2002 cowpea planting seasons. Three bands, each representing a block and running perpendicular to the river, were demarcated, about 150 m apart. Five fields at 200 m-intervals were located along each band. The fields were planted with Tchawé kpayo, the local cowpea cultivar, cultivated and weeded by farmers using traditional cultural practices. In each field, a plot of 10 x 20 m was marked, within which six 2 m2 sub-plots were demarcated. All cowpea plants within each sub-plot were examined and the number of plants showing symptoms of damping-off or stem rot were recorded and tape marked at seven, 15 and 30 d after planting. Disease assessment of these two disease syndromes was expressed as disease incidence. Two plants with the disease symptoms were randomly selected and dug from each 2 m2 sub-plots. Shoots were excised and hypocotyls or stems were collected and transported to the laboratory for fungal pathogen isolation and identification.
Isolations
Diseased stem tissue was cut into small segments (± 2 mm2), surface disinfected with 0.5 % sodium hypochlorite (NaOCl) for 15 s, rinsed twice in SDW, blotted dry on sterile tissue paper and plated onto potato dextrose agar (PDA) amended with 0.025 % chloramphenicol. After 2 d’ incubation at 25±1 °C, pure cultures were obtained by transferring fungal colonies to new PDA and water agar (WA) plates and incubating at 25±1 °C under fluorescent light for five to 10 d. The pure fungal cultures were maintained on PDA slants at 4 °C.
Fungal colony morphological characteristics
The radial growth of the colonies was measured after 3 d incubation, and the colony colour and number of produced sclerotia were recorded. Isolate identification was done using the Mycology Guidebook (Stevens, 1974) based on colony morphology, growth, spores, conidia and sclerotia size and shape. The system of Nelson et al. (1983) was also followed for the identification of some of the fungi whereas the keys of Domsch et al. (1980) were used for the identification of others. One pure culture was deposited in the National Collection of Fungi, Biosystematics Division, Plant Protection Research Institute, Pretoria (PREM 57252) and the remainder in the Collection of Fungi, Plant Health Management Division, International Institute of Tropical Agriculture, Cotonou, Benin (isolate codes: IITA 407, IITA 408, IITA 409, IITA 410, IITA 411, IITA 412).
Pathogenicity tests
Pathogenicity of each isolate was tested in the greenhouse at the International Institute of Tropical Agriculture (IITA), Cotonou Station, Republic of Benin. Soil inoculation was done as described earlier, in Chapter 3.
Cowpea seeds of the Tchawé kpayo cultivar were obtained from the Ouémé valley village. The seeds were surface disinfected in 1 % NaOCl for 2 min, rinsed twice in SDW and then planted in pots (14 cm diam. x 18 cm height) filled with isolate-inoculated soil. There were four seeds per pot, three pots per isolate and repeated four times. The pots were. Pots were kept at 21-30 °C in the greenhouse, watered every 2 d and the number of seedlings showing symptoms of damping-off and stem rot in each pot were recorded at seven and 30 d after planting. Koch’s postulates were fulfilled as described in Chapter 3.
Effect of in vivo interactions among F. oxysporum, F. scirpi, S. rolfsii and T. harzianum on damping-off and stem rot of cowpea in the greenhouse
The in vivo interaction experiment was carried out in the greenhouse to test the synergistic or antagonistic effect of the different fungal species on cowpea using the technique of millet seed inoculum (Weideman and Wehner, 1993), described in Chapter 3. The experimental design was a randomised block design with four replicates of 19 treatments (Table 4.1). Tchawé kpayo cultivar seeds were surface disinfected and planted in pots as described earlier. The number of damping-off seedlings was recorded 3 d after planting and at 2-d intervals thereafter until 30 d after planting.
Field evaluation of the effects of environmental conditions on disease development
On-farm trials were conducted during 2001 and 2002 in a village (Agonguey) in the Ouémé valley to determine the effect of environmental conditions (rainfall, air temperature, relative humidity and soil moisture) and natural field initial inoculum density on disease incidence in cowpea. The village is transected by a permanent river that, each year, toward the end of the rainy season (August-September), overflows and floods the fields of the village. Cowpea is grown after the drainage of the water from the fields. The fields are known to have a history of damping-off and stem rot of cowpea (PEDUNE-BENIN, 1995). During the 2001 cowpea planting season experiment, three bands were demarcated, planted with Tchawé kpayo, and the cultural practices were as described earlier. The experimental design was a complete block with each band representing a block, replicated three times along the river. A 1.0 kg soil sample from a depth of 20 cm was taken at planting from each subplot for initial inoculum density determination. The wet-sieving method described by Punja et al. (1985) was used to directly recover and enumerate sclerotia from the soil samples. Soil moisture was measured gravimetrically from a 20 cm-deep soil sample, taken per subplot at planting and at 7-d intervals until harvest, using the technique of Nielsen et al. (1995). Seven d after planting and subsequently at 7-d intervals until harvest, all cowpea plants within each sub-plot were examined and the number of seedlings or plants showing symptoms of damping-off or stem rot recorded and tape marked to establish disease incidence. Rainfall was recorded daily using a funnel-type gauge positioned 1.5 m above the soil surface in the field. The air temperature and relative humidity were measured daily in the field for the duration of the experiment using a hygrothermograph placed at a height of 30 cm above the cowpea plant canopy. To ensure measurement accuracy, the instrument was housed in a Stevenson’s screen and protected from direct sunlight and rain.
In the second experiment during the 2002 cowpea planting season, three cowpea cultivars Tchawé kpayo, Tchawé daxo and IT83D-326-2 were planted per block in the field. Tchawé kpayo is a susceptible cultivar and Tchawé daxo is a tolerant cultivar being grown by farmers in the valley whereas IT83D-326-2 is known to be moderately tolerant to damping-off and stem rot (PEDUNE-BENIN, 1995). The experimental fields were located along the bands demarcated along the river as described above and each cultivar was separately grown in a 10 x 20 m-plot (per field). The cultural practices, sampling and collection of environmental data were as described earlier.
CHAPTER 1 GENERAL INTRODUCTION
1. 1 Literature cited
CHAPTER 2 LITERATURE REVIEW
2.1 Cowpea: Taxonomy, origin and growth habit
2.2 Worldwide cowpea production
2.3 Cowpea in the Republic of Benin
2.4 Importance of cowpea
2.5 Cowpea damping-off and stem rot-causing fungi
2.6 Sclerotium rolfsii: Nomenclature, host range and distribution
2.7 Sclerotium rolfsii: host tissue infection and pathogenicity
2.8 Sclerotium rolfsii: Epidemiology
2.9 Sclerotium rolfsii: Sexuality and genetics
2.10 Sclerotium rolfsii control measures to protect cowpea from damping-off and stem rot
2.11 Literature cited
CHAPTER 3 OCCURRENCE AND DISTRIBUTION OF COWPEA DAMPING-OFF AND STEM ROT AND ASSOCIATED FUNGI IN BENIN
3.1 Introduction
3.2 Materials and Methods
3.3 Results
3.4 Discussion
3.5 Acknowledgments
3.6 Literature cited
CHAPTER 4 ETIOLOGY OF AND EFFECT OF ENVIRONMENTAL FACTORS ON DAMPING-OFF AND STEM ROT OF COWPEA IN OUÉMÉ, BENIN
4.1 Introduction
4.2 Materials and Methods
4.3 Results
4.4 Discussion
4.5 Acknowledgements
4.6 Literature cited
CHAPTER 5 GENETIC VARIATION AMONG SCLEROTIUM ISOLATES FROM BENIN, DETERMINED USING MYCELIAL COMPATIBILITY AND ITS rDNA SEQUENCE DATA 70
5.1 Introduction
5.2 Materials and methods
5.3 Results
5.4 Discussion
5.5 Acknowledgments
5.6 Literature cited
CHAPTER 6 A NEW LABORATORY TECHNIQUE FOR RAPID SCREENING OF COWPEA CULTIVARS FOR RESISTANCE TO DAMPING-OFF AND STEM ROT CAUSED BY SCLEROTIUM ROLFSII
6.1 Introduction
6.2 Materials and Methods
6.3 Results
6.4 Discussion
6.5 Acknowledgements
6.6 Literature cited
CHAPTER 7 PHENOLIC CONTENT IN COWPEA AS A SCREENING PARAMETER FOR RESISTANCE OR SUSCEPTIBLE TO DAMPING-OFF AND STEM ROT CAUSED BY SCLEROTIUM ROLFSII
7.1 Introduction
7.2 Materials and Methods
7.3 Results
7.4 Discussion
7.5 Acknowledgements
7.6 Literature cited
CHAPTER 8 EFFICACY OF MORINGA OLEIFERA LEAF EXTRACTS AGAINST SCLEROTIUM ROLFSII AND DAMPING-OFF AND STEM ROT OF COWPEA
8.1 Introduction
8.2 Materials and Methods
8.3 Results
8.4 Discussion
8.5 Acknowledgements
8.6 Literature cited
CHAPTER 9 BIOCONTROL AGENTS IN COMBINATION WITH MORINGA OLEIFERA EXTRACT FOR INTEGRATED CONTROL OF SCLEROTIUM-CAUSED COWPEA DAMPING-OFF AND STEM ROT
9.1 Introduction
9.2 Material and methods
9.3 Results
9.4 Discussion
9.5 Acknowledgements
9.6 Literature cited
CHAPTER 10 GENERAL DISCUSSION
10.1 Literature cited
SUMMARY
