Ecology and Management of Charcoal Rot (Macrophomina Phaseolina) on Cowpea in the Sahel

Ecology and Management of Charcoal Rot (Macrophomina phaseolina) on Cowpea in the Sahel

Promotor: Prof. Dr. Ir. A.H.C. van Bruggen Hoogleraar Biologische Landbouwsystemen

Co-promotor: Dr. Ir. A.J. Termorshuizen Universitair Hoofddocent, leerstoelgroep Biologische Landbouwsystemen

Promotiecommissie: Prof. Dr. M.J. Jeger, Imperial College London, Kent, United Kingdom Prof. Dr. Ir. P.C. Struik, Wageningen Universiteit Prof. Dr. L. Brussaard, Wageningen Universiteit Prof. Dr. P.W. Crous, Centraalbureau voor Schimmelcultures, Utrecht

Dit onderzoek is uitgevoerd binnen de onderzoekschool Production Ecology and Resource Conservation

Ecology and Management of Charcoal Rot (Macrophomina phaseolina) on Cowpea in the Sahel

Mbaye Ndiaye

Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit Prof. Dr. M.J. Kropff in het openbaar te verdedigen op vrijdag 15 juni 2007 des namiddags te 16.00 uur in de Aula

Bibliographic data

Mbaye Ndiaye (2007) Ecology and management of charcoal rot (Macrophomina phaseolina) on cowpea in the Sahel PhD Thesis Wageningen University, the Netherlands With summaries in English, French and Dutch x + 114 pp.

ISBN 978-90-8504-664-6 PREFACE

Established in 1974, the regional training and application in Agrometeorology and operational Hydrology (AGRHYMET, Niamey, Niger) is a specialised institution of CILSS (Inter-State Committee on Drought Control in the Sahel). CILSS is composed of nine member countries: Burkina-Faso, Cape Verde, Chad, Gambia, Guinea-Bissau, Mali, Mauritania, Niger and Senegal. The overall goal of the Centre AGRHYMET is to contribute to achieving food security and increased agricultural production in the CILSS member States by providing training in agrometeorology, hydrology and crop protection, producing and disseminating information to development stakeholders and partners. The Department of Training and Research (former Department for Crop protection) was founded in collaboration with Wageningen University and Research Centre. Wageningen University contributed to capacity building for teaching crop protection and for research in the Sahel by giving lectures, and providing agricultural information and technical support for DFR’ trainers. This PhD study is keeping with this collaboration and aimed to develop technologies for charcoal rot control in the Sahel. As I complete this study I would like to thank Wageningen University for offering me a 6-month fellowship and AGRHYMET for funding the research activities in Niger and Senegal. I wish to express my gratitude to Mr Sankung Sagnia, former director of the Department of Training and Research, AGRHYMET, Prof. Arnold van Huis, coordinator WAU-DFPV, Dr. Frans Meerman, backstopping coordinator WAU- DFPV, and Prof. Mike Jeger who offered me possibilities to prepare a PhD thesis. I would like to acknowledge also Mr. Faustin Gnoumou and Mr. Etienne Sarr in charge of the Department of Training and Research and the Pedagogic coordination unit, AGRHYMET respectively for funding the air ticket for my second stay in the Netherlands and for advice. I am grateful to my supervisor Prof. Dr. Ariena van Bruggen for her guidance, encouragements along this work and particularly for thoroughly and consistently reviewing all chapters of this thesis. I am indebted to my supervisor Dr. Aad Termorshuizen for providing suggestions and comments for statistical data analysis and presentation, for guiding the writing of the thesis and for his availability. I also want to thank Dr. Ewald Groenewald and Prof. Dr. Pedro Crous for their contributions to chapter 2. I will not forget to appreciate, the friendship of the staff of the Biological Farming Systems Group of the Wageningen University, where I spent six months. Finally, I am also very grateful for the contributions Mrs. Wampie van Schouwenburg, Mr. Hennie Halm and Dr. Wim Blok made to this thesis.

Contents

CHAPTER 1 General introduction 1

CHAPTER 2 Physiological, genetic, and pathogenetic variability in Macrophomina phaseolina, the causal agent of charcoal rot 25

CHAPTER 3 Effect of rotation of cowpea (Vigna unguiculata) with Digitaria exilis and Pennisetum glaucum on Macrophomina phaseolina densities and cowpea yield 47

CHAPTER 4 Effects of compost amendment on charcoal rot (Macrophomina phaseolina) development of cowpea 61

CHAPTER 5 Combined effects of solarization and organic amendment on charcoal rot caused by Macrophomina phaseolina in the Sahel 79

CHAPTER 6 General discussion 91

Summary 97 Résumé 101 Samenvatting 105 Publications of the author 109 Curriculum vitae 111 PE&RC PhD Education Certificate 113

CHAPTER 1

General introduction

The Sahel is a large area bordering the Sahara desert to the South. In West Africa, it covers the larger part of Burkina Faso, Cape Verde, the Gambia, Guinea Bissau, Mali, Mauritania, Niger, Senegal, and Chad. In these regions, the climate is semi-arid, which is defined by the precipitation being less than potential evaporation. The region lies between 200–600 mm annual rainfall isohyets with erratic rains falling during 3–5 months per year and occurrence of drought spells during the period of crop growth. Temperatures during the hottest months of the dry season are high (30–45°C) (Morrel, 1992). Consequently, the vegetation of the Sahel region, composed mainly of sparse trees, shrubs, bushes and grasses, has a restricted development (Penning de Vries and Djitèye, 1982). This condition of low biomass production associated with subsistence cultivation and animal overgrazing has contributed to a decreasing organic matter content of the Sahelian soils. As a result, only few crops can be grown successfully and among them cowpea and millet are the more widespread. Especially the rapid growth of cowpea, associated with high nutritional quality and potential yields in conditions of limited inputs, has made this crop a widely grown food and cash crop in the Sahel.

Importance of cowpea in the Sahel Cowpea (Vigna unguiculata (L.) Walp.) is a valuable source of protein for human and animal nutrition. It is mainly grown to produce dry grains, but about 25% is consumed on-farm or marketed as green pods. The grain and fresh peas have a high protein content (about 25% on a dry weight basis) and an amino acid profile that complements the proteins present in rice, millet, maize or wheat. Thus, a combination of 75% cereal and 25% cowpea diet, on a dry weight basis, provides sufficient proteins for adults (Cisse and Hall, 2002). Besides, cowpea hay has a higher protein content than grass hay and thus constitutes a valuable feed for livestock (Tarawali et al., 1997). Cowpea roots have Rhizobium root nodules that are able to fix nitrogen even in very poor soils (organic matter less than 0.2%, sand content > 85%). Cowpea is shade-tolerant and therefore compatible as an intercrop with a number of crops (Singh and Sharma, 1996). All these qualities have contributed to the widespread distribution of cowpea in the semi-arid regions of West Africa, where other food legumes do not perform well.

1 Chapter 1

Cowpea production and constraints in the Sahelian zone The main economic activity in the Sahelian countries comprises agriculture, employing 70% of the population. However, the agricultural income per person is very low (US $ 60–140 per year) due mainly to climatic constraints (CILSS/PRORES, 1997). Conditions of drought, resulting in water deficit and stress are common in these areas. The reduction of the rains in the last decades has influenced the duration of the cropping season, such that formerly cultivated varieties no longer can complete their development cycle. Variation in rainfall further hampers the agricultural production. Often water deficit occurs in August to such an extent that cowpea, which flowers during that period, is negatively affected. Cowpea is the dominant staple pulse crop in Niger and Senegal (FAOSTAT, 2005) and is usually intercropped with pearl millet (Pennisetum glaucum) in Niger or cultivated as sole crop in Senegal. In both countries, cowpea-based subsistence cropping systems receive few if any inputs of fertilizer or pesticides and are cultivated often without rotation (millet/cowpea intercropping or cowpea sole crop) or in biannual rotation with millet. Satisfactory nodulation is naturally obtained by indigenous Rhizobium strains that can fully supply the needs for nitrogen. However, nitrogen fixation only begins to become effective about three weeks after planting. Therefore, an application of 9 kg N/ha just prior to planting is required in infertile Sahelian soils to satisfy the need of nitrogen of the seedlings (Cisse et al., 1995). The advances made in breeding cowpea since 1984 by the International Institute of Tropical Agriculture (IITA) and mandated National Research Institutes have led to a release of various cowpea cultivars that perform well in sole cropping and intercropping (Singh, 1994). In Senegal, new cowpea varieties have been released with a cultivation period of 60-64 days that have produced yields as high as 1 ton/ha in the Louga Region in years with only 200 mm of rain (Cisse et al., 1995; Cisse et al., 1997). These varieties are resistant to bacterial blight and most cowpea aphid-borne viruses present in Senegal. Unfortunately, they are susceptible to the soilborne fungal pathogen Macrophomina phaseolina. Damage inflicted by this pathogen is becoming more frequent and severe, because of cowpea monoculture and erratic rainfall in the cowpea production areas. Although achievements made by breeding new cowpea varieties suitable for intercrop and sole crop and drought conditions, average yields in farmers' fields in the Sahel remain very low (0.2–0.5 ton/ha). Charcoal rot, which affects the water balance and plant turgor, is one of the most important constraints to the production (Emechebe and Shoyinka, 1985; Adam, 1986; 1990; Paré, 1990).

2 General introduction

Macrophomina phaseolina

Taxonomy and nomenclature Macrophomina phaseolina (Tassi) Goid. (= Tiarosporella phaseolina (Tassi) Van der Aa) is a soilborne plant pathogenic fungus. It belongs to the anamorphic Ascomycetes and is characterized by the production of both pycnidia and sclerotia in host tissues and culture media. The pycnidial state was initially named Macrophoma phaseolina by Tassi in 1901 and Macrophoma phaseoli by Maublanc in 1905. In 1927, Ashby maintained the name Macrophomina phaseoli, while Goidanich (1947) proposed Macrophomina phaseolina. Tiarosporella phaseolina (Tassi) Van der Aa was used in 1981 by Van der Aa to designate the species. Mihail (1992) indicated that there is an unconfirmed report of a teleomorph named Orbilia obscura (Ghosh et al., 1964) of M. phaseolina, but since then no further evidence appeared for the telemorph state. The sclerotial state was described for the first time by Halsted as Rhizoctonia bataticola (Taub.) Butler on Ipomoea batatas in 1890. According to Dhingra and Sinclair (1978), the same fungus was isolated from cowpea in India in 1912 by Shaw and was then named Sclerotium bataticola. Recently Crous et al. (2006) demonstrated that although the telemorph is unknown, M. phaseolina is a member of the family Botryosphaeriaceae. The authors pointed out the differences between Tiarosporella and Macrophomina, which produces in the pycnidia percurrently proliferating conidiogenous cells. The pycnidiospores are ellipsoid to obovoid, and measure (16-)20-24(-32) × (6-)7-9(-11) µm. During the sclerotial formation, 50–200 individual hyphal cells aggregate to give multicellular bodies named microsclerotia. The microsclerotia are black and are variable in size (50–150 µm) depending on the available nutrients of the substrate on which the propagules are produced (Short and Wyllie, 1978).

Isolates and pathogen populations Much work has been done to elucidate the variability in morphology, physiology, pathogenicity, and genotype of M. phaseolina. Variation in cultural characteristics and virulence to soybean has been reported in the U.S. (Dhingra and Sinclair, 1973). According to Ahmed and Ahmed (1969), cultural characteristics and growth rates of 8 different jute isolates of M. phaseolina appeared to be related to their pathogenicity. Isolates with fast mycelial growth and abundant sclerotial production were more pathogenic on clusterbean (Cyamopsis tetragonoloba) seedlings than isolates growing more slowly (Purkayastha et al., 2004). Color of cultures on PDA, ability to sporulate in infected host plants and pycnidial size also have been reported to vary greatly (Dhingra and Sinclair, 1978; Adam, 1986). Isolates of M. phaseolina obtained from

3 Chapter 1 resistant sorghum genotypes were more pathogenic on susceptible sorghum than two other isolates originally obtained from susceptible sorghum genotypes (Diourte et al., 1995). Tentative demonstration of host preference of M. phaseolina isolates was done by Pearson et al. (1986; 1987a, b). Comparing growth aptitude of more than 2000 isolates from 13 states in medium containing potassium chlorate, the authors classified isolates of M. phaseolina from maize as chlorate-resistant and those from soybean as chlorate-sensitive. The growth of the last group was inhibited by chlorite produced in the medium. In contrast, Zazzerni and Tosi (1989) reported that M. phaseolina isolates from four host species varied widely in chlorate-utilization irrespective of their original host and concluded that there was no evidence for host specialization within M. phaseolina. Further evidence of lack of host specialization in M. phaseolina was reported by Mihail and Taylor (1995). Their study on the variability of 114 M. phaseolina isolates from different host species, soils and continents clearly indicated that M. phaseolina is a heterogeneous species that cannot be partitioned into distinct subspecies groups based on pathogenicity, pycnidium production and chlorate utilization. Although Su et al. (2001) pointed out that host specialization in M. phaseolina occurs with maize, no clear evidence for the occurrence of formae speciales, subspecies or physiological races has been reported so far. Various recent studies were devoted to the genetic and pathogenic variability of M. phaseolina. Significant advances in molecular detection and differentiation of M. phaseolina isolates have been achieved using Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), and Amplified Fragment Length Polymorphism (AFLP) analysis (Mayek-Pérez et al., 2001; Su et al., 2001; Purkayastha et al., 2006; Reyes-Franco et al., 2006). So far, none of these methods have however been able to differentiate isolates of M. phaseolina from specific hosts or geographic locations. The lack of a strong correlation between genotype and geographical origin suggests a high diversity level within and among the population of M. phaseolina (Jana et al., 2005). Nevertheless, Jana et al. (2003) developed a single RAPD primer OPA-13 that distinguishes isolates of M. phaseolina from soybean, sesame, groundnut, chickpea, cotton, common bean, okra, and 13 other hosts and this could be useful as taxonomic marker for population studies.

Disease cycle M. phaseolina causes seedling blight, root rot and root and stem rot of more than 500 cultivated and wild plant species including economically important crops as soybean, common bean, sorghum, maize, cotton, peanut, cowpea (Dhingra and Sinclair, 1977; Bouhot, 1967, 1968; Adam, 1986; Gray et al., 1990; Hall, 1991; Diourte et al., 1995). Softwood forest trees (Abies, Pinus, Pseudotsuga) (McCain and Scharpf, 1989), fruit

4 General introduction trees (Citrus spp., Cocoa nucifera, Coffea spp.) and weed species (Songa and Hillocks, 1996) are also hosts of the pathogen. The fungus was reported in North and South America, Asia, Africa and Europe. However, it is economically more important in subtropical and tropical countries with a semi-arid climate (Wrather et al., 1997; 2001). M. phaseolina produces sclerotia in root and stem tissues of its hosts which enable it to survive adverse environmental conditions (Cook et al., 1973; Meyer et al., 1974; Short et al., 1980). In PDA, pycnidia are not produced except under some specific incubation conditions (Gaetán et al., 2006) and only sometimes in host crops (Mihail and Taylor, 1995), and their importance in the epidemiology of the fungus likely depends on the host involved as well as the fungal isolate (Ahmed and Ahmed, 1969). On cowpea, pycnidia are produced at the end of the rainy season, but their epidemiological significance seems minor. On the contrary, in jute crops, pycnidiospores produced on early infected stem and leaf tissues have been reported to be responsible for secondary spread of the disease (Anonymous, 1940). Microsclerotia in soil, infected seeds or host tissues serve as primary inoculum (Bouhot, 1968; Dhingra and Sinclair, 1977; Abawi and Pastor-Corrales, 1990). Root exudates induce germination of microsclerotia and root infection of hosts. The infective hyphae enter into the plant through root epidermal cells or wounds. During the initial stages of pathogenesis, the mycelium penetrates the root epidermis and is restricted primarily to the intercellular spaces of the cortex of the primary roots. As a result, adjacent cells collapse and heavily infected plantlets may die. At flower onset, the fungal hyphae grow intracellularly through the xylem and form microsclerotia that plug the vessels (Short et al., 1978; Mayek-Pérez et al., 2002) and disrupt host cells. The infected plants show necrotic lesions on stems, branches, and peduncles. From pod peduncles, the fungus spreads to the pods and invades grains. Heavily infected plants die prematurely due to the production of fungal toxins e.g. phaseolinone (Bhattacharya et al., 1994) and production of fungal tissue that plugs host vessels. In soybean, formation of microsclerotia is conditioned by flowering and pod setting (Wyllie and Calvert, 1969) and may be indicative of initiation of death of the host (Short and Wyllie, 1978). After plant death, colonization by mycelia and formation of sclerotia in host tissue continue until tissues are dry. The mycelium and microsclerotia produced in infected plant material, including plant residues are the means of propagation of the pathogen. Microsclerotia in soil, host root and stems are the main surviving propagules. After decay of root and plant debris, microsclerotia are released into the soil. They are distributed generally in clusters at the soil surface and are localized mainly at a depth of 0–20 cm (Alabouvette, 1976; Mihail, 1989; Campbell and van der Gaag, 1993). They can survive for 2–15 years depending on environmental conditions, and whether or not the sclerotia are associated with host

5 Chapter 1 residue (Cook et al., 1973; Papavizas, 1977; Short et al., 1980; Baird et al., 2003). Factors that adversely affect the survival of these propagules include repeated freezing and thawing of soil, low carbon:nitrogen ratios in soil, and soil moisture content (Dhingra and Sinclair, 1974; Dhingra and Sinclair, 1975).

Symptoms on cowpea On cowpea, disease symptoms are clearly visible from the time of emergence and can be evaluated at various stages of development of the plant. After emergence, symptoms can be observed on the cotyledons as brown to dark spots. However, cotyledons remain on the plant for only a few days, especially when diseased. The margins of the cotyledons become bright red, then beige or brown, and finally brown to black. Often, they are covered with a grayish mycelium pad bearing scattered sclerotia. This mycelium can be observed also inside entirely colonized cotyledons. At the unifoliate leaf stage, typical symptoms are pinhead-size, charcoal-colored spots which are mostly restricted to the hypocotyl section of the stem, including its subterranean part. Infected spots may expand and develop into large necrotic lesions, usually resulting in death of the plant. M. phaseolina can also infect roots which show necrotic lesions (Bouhot, 1967; Adam, 1986). On adult plants, M. phaseolina causes lesions on stems, spikes, pods and seeds. On stems, lesions are beige and appear at the ramification point of the lateral secondary branches. Colonized tissues become gray and covered with abundant minute black punctuations. Initially these punctuations are immersed, becoming gradually more prominent. From pod peduncles, the fungus spreads to the pods and invades grains. However, necrotic lesions may appear anywhere on the pods. Infected green pods are initially blue-green, and then turn brown to reddish. When infection occurs on mature, dry pods, they become white to gray and are covered with locally or widely distributed black bodies on the pod. The fungus penetrates the pod and grains, inducing diverse symptoms depending on tissue infestation level. Early infested grains abort or become empty and dry. The affected parts of the pod become narrow or shrunken, deformed and thin when damaged grains are located at the pod tip (Bouhot, 1965; 1967). The most striking symptom is the sudden wilting and drying of the whole plant, most of the leaves remaining green. The stem and branches are then covered with black bodies and give the charcoal or ashy appearance of dead plants. Withering can be observed from seedling to maturing stage and is the result of necrosis of roots, stems and mechanical plugging of xylem vessels by microsclerotia, but also by toxin production, and enzymatic action (Chan and Sackston, 1973; Kuti et al., 1997; Jones and Wang, 1997).

6 General introduction

Factors affecting the infection and severity of the charcoal rot disease Root infection is affected by growth stage and environment. High root infection can occur before reproductive development and is then associated with hot and dry weather early in the growing season (Cloud and Rupe, 1994). M. phaseolina can infect beans also under relatively dry conditions (Olaya and Abawi, 1993). However, there are also reports where a high moisture holding capacity (40–50% MHC) resulted in greater M. phaseolina colonization on peanut (Husain and Ghaffar, 1995). Agarwal and Goswani (1973) reported a significant synergistic effect in soybean when the root- knot nematode Meloidogyne incognita preceded infection by M. phaseolina by three weeks, and suggested that M. incognita predisposes plants to the fungal infection, similar to the vascular pathogens Fusarium oxysporum and Verticillium dahliae. In white clover, M. phaseolina also tends to be associated with higher final densities of the plant pathogenic nematodes Meloidogyne trifoliophila, Helicotylenchus dihystera and Heterodera trifolii (Zahid et al., 2002). In contrast, in a pot experiment the simultaneous addition of M. phsaeolina and Meloidogyne javanica resulted in reduced nematode galls, which was ascribed to the effect of toxic metabolites on the nematode produced by the fungus (Gupta and Mehta, 1989). Many studies have demonstrated the lack of consistent correlation between the severity of host infection and charcoal rot incidence. Visible symptoms of the disease in the field are most apparent under conditions that reduce plant vigor, such as poor soil fertility (Sinclair and Backman, 1989), high seeding rates (Pearson et al., 1984; Sinclair and Backman, 1989), low soil water content (Meyer et al., 1974; Ali and Ghaffar, 1991; Sheikh and Ghaffar, 1979; Kendig et al., 2000), high temperatures (Odvody and Dunkle, 1979; Mihail, 1989), and root injury (Canaday et al., 1986). The timing of host reproduction is another factor that has a strong influence on charcoal rot development. In Euphorbia lathyris, early flowering plants succumb more rapidly to charcoal rot than later flowering ones (Mihail, 1989). In sorghum, post-flowering water-stressed plants showed more severe charcoal rot symptoms than plants without water stress (Diourte et al., 1995). Initial population density of sclerotia in soil was directly correlated with the severity of charcoal rot of soybean and was inversely related to soybean yield (Short et al., 1980). Mihail (1989) found that average symptom expression and mortality increased with increasing soil temperature and that mortality increased markedly after the soil temperature at 5 cm reached the range of 28–30°C.

Management strategies Most of the described control methods aim to reduce the number of sclerotia in soil or to minimize the contact of the inoculum and the host. Solarization (Grinstein et al.,

7 Chapter 1

1979; Katan et al., 1980; Pullman et al., 1981; Usmani and Ghaffar, 1982), addition of organic amendments (Ghaffar et al., 1969; Dhingra and Sinclair, 1975), maintenance of high soil moisture content (Dhingra and Sinclair, 1975) and fumigation (Watanabe et al., 1970) have been suggested as possible methods to manage soilborne pathogens. Solarization alone was not effective at controlling M. phaseolina in forest (Old, 1981) and field (Mihail and Alcorn, 1984) soils. Soil moisture content greatly affects the sensitivity of resting structures to heat treatment (Lodha et al., 2003), and one summer irrigation was sufficient to reduce the population of M. phaseolina by 25–42% (Lodha and Solanki, 1992; Lodha, 1995). Solarization of moistened soil further augmented this reduction in the top soil, but many propagules survived at lower depths (Lodha and Solanki, 1992). Amendments with nitrogen-enriched pearl millet residues significantly reduced the population of M. phaseolina within 45 days by 94% (Sharma et al., 1995). Combined effects of amendments, irrigation and polyethylene mulching resulted in the almost complete eradication of the population of M. phaseolina (93– 99% reduction) at 0–30 cm soil depth within 15 days. A considerable reduction (75– 95%) was also achieved by natural heating of irrigated soil for 15 days after amending with cruciferous residues (Lodha et al., 1997). Cabbage residue incorporation in the soil without heating also reduced the population of Fusarium oxysporum f. sp. conglutinans effectively (Ramirez-Villapudua and Munneke, 1987). The effect was mainly attributed to toxic volatiles such as mercaptan, dimethyl sulphid, and isothiocyanate formed during the decomposition of the cabbage residues (Gamliel and Stapleton, 1993). However, crucifer crops are not grown in the Sahel. Tillage is a crucial cultural measure that could affect the inoculum potential of soilborne pathogens. If the pathogen requires high inoculum density to infect plants, then increased dispersal over the soil profile could reduce disease severity. If, however, a low inoculum density is sufficient for infection, then dispersal may aggravate incidence and severity (Olanya and Campbell, 1988). As low inoculum densities are sufficient to cause charcoal rot, tillage can increase damage by M. phaseolina especially when highly susceptible hosts such as Euphorbia lathyris are cultivated, in which a soil sclerotial density < 1 microsclerotium per gram soil can cause more than 90% plant mortality (Young and Alcorn, 1984). Tillage reduces the stratification of organic residue on the surface, which in turn can influence soil temperature and moisture (Campbell and van der Gaag, 1993), soil chemistry (Blevins et al., 1980), population of soil animals, and the structure of microbial communities (Franchini et al., 2006). These changes in physical and biological factors may in turn also affect disease incidence and severity of M. phaseolina. Irrigation at any time during the cropping season reduces disease infection in soybean (Kendig et al., 2000). Furthermore the type of irrigation can also affect

8 General introduction charcoal rot disease. The density of soil sclerotia and the number of diseased melon plants was higher in drip irrigated plots than in furrow irrigated plots (Nischwitz et al., 2004). Sources of resistance to some soilborne pathogens have been identified, but highly resistant cultivars are often not available for polyphagous and unspecialized pathogens like M. phaseolina. Pastor-Corrales and Abawi (1988) reported some selected bean lines with stable resistance to the fungus. Resistance in beans to M. phaseolina has been associated with drought tolerance (Pastor-Corrales and Abawi, 1988). In common bean cv. BAT-477 resistance to M. phaseolina is controlled by two dominant complementary genes (Olaya et al., 1996). Grezes-Besset et al. (1996) reported resistance to M. phaseolina in Ricinus persicus and incorporated this in cultivated castor bean. The improved lines showed high seedling resistance to the disease. Based on seed yield and the levels of lower stem and taproot colonization, Smith and Carvil (1997) identified four resistant cultivars among 24 soybean cultivars screened for resistance to M. phaseolina. Research on sources for resistance to M. phaseolina in sorghum has led to breeding lines and cultivars with stable performance. Resistance in sorghum was associated with delayed leaf senescence (Ducan, 1984; Diourte et al., 1995). The stability of this resistance, however, was influenced by water stress. In soybean, resistance factors to M. phaseolina do not protect plants against infection, but more likely restrict the growth rate of the fungus within plant tissues (Smith and Carvil, 1997). Reduced growth of the pathogen within host tissues may be due to lower levels of the stress related free amino acids proline and asparagine in resistant than in susceptible cultivars (Pearson et al., 1987b). Cowpea cultivars highly resistant to M. phaseolina and adapted to the different production areas with acceptable agronomic characteristics are not available. Moderate levels of resistance were reported in India (Sohi and Rawal, 1983; Singh and Lodha, 1986; Mahabeer et al., 1995). In Niger and Senegal several cowpea varieties and lines were screened for resistance to M. phaseolina in a disease nursery and pot experiments. These accessions including lines with delayed leaf senescence and drought resistant cultivars were susceptible to the pathogen (Adam, 1986; M. Ndiaye unpublished). Nevertheless, better cowpea stands were attributed to moderate resistance of one cultivar (Gaikwad and Sokhi, 1987). Higuera and Murty (1987) screened 414 cowpea progenies of white and black seed types in a study to verify the supposed resistance to M. phaseolina in cowpea black seed types. The authors concluded that seed color had no influence on resistance, but that significant variation in resistance to charcoal rot exists among cowpea accessions which could be used in breeding programs. Several studies have dealt with the antifungal effect of plant compounds on M.

9 Chapter 1 phaseolina. The essential oil actinidine isolated from Nepeta clarkei was effective in vitro against M. phaseolina (Saxena and Matela, 1997). Kazmi et al. (1995) and Alice et al. (1996) reported that in vitro neem oil was more or equally effective compared to benomyl and carbendazim. However, neem extracted from seed samples of different localities showed variable suppression of growth of the pathogen. More effective inhibition of the growth of M. phaseolina was obtained by aqueous extracts of Cymbogon citratus (Bankole and Adebanjo, 1995). Powder of Datura fastulosa (datura) was also reported to be effective against M. phaseolina and Meloidogyne javanica infection in a pot experiment (Enteshamul et al., 1996). Management strategies to control charcoal rot also include the use of biocontrol agents to prevent host infection or to suppress the growth of the pathogen (Ghaffar et al., 1969; Siddiqui and Mahmood, 1993). In jasmine, Trichoderma harzianum and T. viride were effective against M. phaseolina (Alice et al., 1996). T. harzianum isolate 25 and Pseudomonas fluorescens isolate 4 significantly reduced the germination of sclerotia by 60% in natural field soil (Srivastava et al., 1996). Strains of Bradyrhizobium sp. and Rhizobium meliloti were reported to be antagonistic against M. phaseolina and to have plant growth promoting properties in groundnut (Arora et al., 2001; Deshwall et al., 2003). Researchers also have investigated the in vitro sensitivity of different isolates of M. phaseolina to fungicides (Al-beldawi et al., 1973; Rama et al., 1981) and the efficacy of fungicide application to seed and soil to reduce fungal germination and infection (Kannaiyan et al., 1980; Alice et al., 1996). However, until now, chemical control of M. phaseolina is difficult and neither profitable nor advisable (Person et al., 1984). This study focuses on cultural and biological control of charcoal rot in cowpea cultivated in the semi-arid Sahelian region of West Africa, because resistance of cowpea against M. phaseolina is lacking.

Problem definition and objectives Although references are scanty, the impression exists that charcoal rot caused by M. phaseolina has progressed fast in the semi-arid zone of the Sahel. Previous studies on M. phaseolina have investigated variation in morphology and pathogenicity among isolates of various hosts (Dhingra and Sinclair, 1973; Echávez-Badel and Perdomo, 1991; Purkayastha et al., 2006). Although only one species is recognized within the genus Macrophomina (Su et al., 2001), Mihail and Taylor (1995) found great variability in pathogenicity among isolates from the Asteraceae, Euphorbiaceae, Fabaceae, and Poaceae. Isolates colonizing the Poaceae were more restricted in pathogenicity than the general population.

10 General introduction

Purkayastha et al. (2006) reported the existence of a high degree of polymorphism in the restricted regions of the internal transcribed spacer region (ITS) and could relate this to chlorate resistance, but not to host species. However, Su et al. (2001) found that M. phaseolina isolates from corn, cotton, and soybean were closely related, although genetic variation could be detected among isolates based on their hosts. Colonization of corn roots was significantly greater with corn isolates than with isolates from other hosts. If host preference among isolates of M. phaseolina exists in Sahelian cropping systems, then certain crop rotation may lead to lower survival of M. phaseolina. Currently no information is available about genetic variation and host specialization for M. phaseolina isolates from the Sahel. Given the environmental constraints in the semi-arid zone of the Sahel, what is the optimal way to manage charcoal rot caused by M. phaseolina? Francl et al. (1988) reported a significant reduction of the populations of M. phaseolina in soil when soybean was rotated with cotton, whilst corn and grain sorghum in rotation with soybean did not lower the density of microsclerotia in soil consistently. In Kenya, under field conditions, the incidence of charcoal rot on common bean was significantly lower after 3 years of monocropping sorghum or maize than after 3-year monocropping of common bean or cowpea (Songa and Hillocks, 1996). These data suggest the possibility to reduce damage by charcoal rot using crop rotation even when moderately susceptible host plants are used. In the Sahel, millet a supposed non-host of M. phaseolina is grown commonly, and after harvest millet stalks are common organic residues. Another cereal crop, fonio (Digitaria exilis), is grown more sporadically because it is labour-demanding in the processing of grains (threshing and husking), but seeds fetch a high price. This crop could be used in the rotation if it would be a non-host. However, fonio has not been tested as rotation crop for control M. phaseolina. A possibility of improving soil fertility and suppressing root disease is through defining proper crop rotations and organic matter management (Alabouvette, 1990; Sharma et al., 1995; Villeneuve, 2000). Compost or manure has been reported to suppress soilborne diseases significantly through increased competitiveness and / or by the presence or stimulation of specific antagonists (Voland and Epstein, 1984; Hoitink and Fahy, 1986). Manure application contributes also to the improvement of soil aggregation. Fungi stabilise soil aggregates, owing to the formation of mycelial filament-soil particle complexes that allow better water and carbon storage (Albrecht et al., 1998), subsequently reducing water stress of plants. Unfortunately, in West Africa, the quantities of animal manure required for soil fertility improvement is not available for the majority of smallholder farmers. One traditional practice used in Burkina Faso by farmers is the so-called Zaï technique, which consists of applying

11 Chapter 1 organic amendments locally, in the seed holes, measuring 25 cm diameter wide and 15 cm deep. Hence, the need of organic matter is then much lower in comparison with the classical broadcasting technique of applying manure (Rose, 1993). The possibility of controlling M. phaseolina with compost in the planting holes has not been investigated thus far. It has been demonstrated that solarization alone is not effective in controlling M. phaseolina in soil (Mihail and Alcorn, 1984). Solarization of moistened soil reduced the resting structures of the fungus in the top soil layer, but inoculum survived at lower soil depths (Lodha and Solanki, 1992). Amendments with nitrogen-enriched pearl millet residues significantly reduced the population of M. phaseolina within 45 days, but not that of Fusarium oxysporum (Sharma et al., 1995). A considerable reduction (75-95%) of the soilborne fungal population was also achieved by natural heating of irrigated soil for 15 days after amending with cruciferous residues (Lodha et al., 2003). However, cruciferous residues are not available in the Sahel, while paunch is commonly available from slaughterhouses in the cities. The management of paunch of slaughtered animals constitutes a real problem in big towns. These materials (fresh or composted) could be used to increase the fertility of the poor sandy soils that characterizes this region and to contribute to the control of soil pathogens. We aimed to investigate the individual and combined effects of soil incorporation of organic amendments available in the region (i.e., paunch of slaughtered animals and millet residues), and solar heating on survival of M. phaseolina and cowpea production. The main goal of the study was to develop tools for management of Macrophomina phaseolina in the Sahel. The objectives were: 1. to characterise cowpea isolates of Macrophomina phaseolina prevalent in the different cowpea cropping systems in Niger and Senegal with respect to culture characteristics and host range; 2. to determine effects of rotation of cowpea with fonio and millet on M. phaseolina disease of cowpea, and production and survival of microsclerotia in soil; 3. to study the effects of compost and a biocontrol agent on M. phaseolina and charcoal rot on cowpea; 4. to study the effects of solarization alone or in combination with organic residues on M. phaseolina and charcoal rot on cowpea.

Outline of the thesis Following this general introduction, the physiological variability and pathogenicity of isolates of M. phaseolina, derived from three cowpea cropping systems of Niger and

12 General introduction

Senegal are presented in Chapter 2. Isolates of M. phaseolina were collected from different hosts and soils. The following characteristics of the isolates of M. phaseolina were determined: in vitro growth rate at various temperatures and pathogenicity to four cowpea cultivars as well as to maize, millet, sorghum, and fonio (Digitaria exilis) in a pot experiment. A subset of isolates was characterized genetically using polymorphism analysis of the ITS region. In Chapter 3, the effect of rotation of cowpea with fonio (Digitaria exilis) and millet (Pennisetum glaucum) on M. phaseolina was studied. The experiment was carried out in a field naturally infested with M. phaseolina. The whole experiment comprised 3 blocks with various levels of soil infestation. The treatments were fonio cropping or millet cropping for 3 years. In the 3rd year, the effects of these treatments on soil inoculum and cowpea production were tested by sowing a susceptible variety of cowpea. Yearly soil inoculum of M. phaseolina and host infection were recorded. In Chapter 4, the effects of composting on survival of M. phaseolina and of compost amendment on disease development and cowpea production were investigated. The effect of composting on viability of the microsclerotia of M. phaseolina was studied by incorporating infected cowpea stems in a compost pit at different depths. Each week, samples were withdrawn from the compost pit and the number of viable sclerotia was determined. To study the disease suppressive effect of the compost, three compost doses were compared to a control in a field naturally infested with M. phaseolina during three seasons. The compost was applied in seed holes. Observations were made weekly from germination to harvest. Incidence, time to death, disease intensity and the area under the disease progress curve (AUDPC) were determined. Germination rate, plant survival, pod, grain and hay dry weight and tissue sclerotial content at physiological maturity of cowpea were also recorded. In a second experiment, the combined effects of compost and local isolates of the fungal antagonist Clonostachys rosea (Gliocladium roseum) on charcoal rot development and cowpea production were determined in the same field. In Chapter 5, we present the results of a study where the combined effects of organic amendment and solar heating for the control of charcoal rot were studied. A field experiment was conducted with a split plot design with solarization as main plot and amendment as subplot. In the main plot, the treatments were solarization and no solarization and in the subplots, control (no amendment), paunch amendment, millet residue amendment, and a combination of paunch and millet residue amendment before solarization. Thirty days after solarization, soil inoculum density of M. phaseolina was determined and cowpea was sown. Disease progress and yield parameters were also determined. Finally, in Chapter 6 all the results of this study are discussed, followed by a general summary.

13 Chapter 1

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15 Chapter 1

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16 General introduction

Franchini, J. C., Crispino, C. C., Souza, R. A., Torres, E. and Hungria, M. 2006. Microbiological parameters as indicators of soil quality under various soil management and crop rotation systems in southern Brazil. Soil Tillage Res. 92: 18–29. Francl, L. J., Wyllie, T. D. and Rosenbrock, S. M. 1988. Influence of crop rotation on population density of Macrophomina phaseolina in soil infested with Heterodera glycines. Plant Dis. 72: 760–764. Gaetán, S. A., Fernandez, L., and Madia, M. 2006. Occurrence of charcoal rot caused by Macrophomina phaseolina on canola in Argentina. Plant Dis. 90: 524. Gaikwad. D. G. and Sokhy, S. S. 1987. Detection of seed rot, root rot and seedling infection in naturally infected cowpea seeds in Senegal and their control. Plant Dis. Res. 2: 127–128. Gamliel, A. and Stapleton, J. J. 1993. Characterization of antifungal compounds evolved from solarized soil amended with cabbage residue. Phytopathology 83: 899–905. Ghaffar, A., Zentmeyer, G. A. and Erwin, D. C. 1969. Effect of organic amendments on severity of Macrophomina root rot of cotton. Phytopathology 59: 1267– 1269. Ghosh, T., Mukherji, N. and Basak, M. 1964. On the occurrence of a new species of Orbilaria Fr. Jute Bull. 27: 134–141. Goidanich, G. 1947. A Revision of the genus Macrophomina Petrak. type species: M. P. (Tassi) G. Goid. n. comb. M. P. (Maubl.) Ashby. Ann. Sper. Agr. N. S. I. 3: 449–461. Gray, F. A., Kolp, B. J. and Mohamed, M. A. 1990. A disease survey of crops grown in the Bay Region of Somalia, East Africa. F. A. O. Plant Prot. Bult. 38: 39–47. Grezes-Besset, B., Lucante, N., Kelechian, V., Dargent, R. and Muller, H. 1996. Evaluation of castor bean resistance to sclerotial wilt disease caused by Macrophomina phaseolina. Plant Dis. 80: 842–846. Grinstein, A., Katan, J., Abdul Razik, A., Zeydan, O., and Elad, Y. 1979. Control of Sclerotium rolfsii and weeds in peanuts by solar heating of the soil. Plant Dis. Rep. 63: 1056–1059. Gupta, D. C. and Mehta, N. 1989. Interaction studies between different levels of Meloidogyne javanica and Rhizoctonia spp. on mung bean (Vigna radiate (L) Wilczek.). Indian J. Nematol. 19: 138–143. Hall, R., ed. 1991. Compendium of bean diseases. American Phytopathological Society, St. Paul, MN. 73 p. Higuera, A. and Murty, B. R. 1987. Response to selection for resistance to Macrophomina and Xanthomonas and its association with seed colour in

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Olaya, G. and Abawi, G. S. and Weeden, N. F. 1996. Inheritance of the resistance to Macrophomina phaseolina and identification of RAPD markers linked to resistance genes in beans. Phytopathology 86: 674–679. Old, K. M. 1981. Solar heating of soil for the control of nursery pathogens of Pinus radiata. Aust. For. Res. 11: 141–147. Papavizas, G. C. 1977. Some factors affecting survival of sclerotia of Macrophomina phaseolina in soil. Soil Biol. Biochem. 9: 337–341. Paré, D. 1990. Technique de quantification de l'inoculum et distribution géographique de Macrophomina phaseolina (Tassi) Goid. au Burkina Faso. Deuxième Séminaire sur la Lutte Intégrée contre les Ennemis des Cultures Vivrières dans le Sahel, 4–9 Janvier 1990. Bamako - Mali. CILSS, INSA, UCTR/PV. 12p. Pastor-Corrales, M. A. and Abawi, G. S. 1988. Reactions of selected bean accessions to infection by Macrophomina phaseolina. Plant Dis. 72: 38–41. Pearson, C. A. S., Leslie, J. F. and Schwenk, F. W. 1986. Variable chlorate resistance in Macrophomina phaseolina from corn, soybean, and soil. Phytopathology 76: 646–649. Pearson, C. A. S., Leslie, J. F. and Schwenk, F. W. 1987a. Host preference correlated with chlorate resistance in Macrophomina phaseolina. Plant Dis. 71: 828-831. Pearson, C. A. S., Leslie, J. F. and Schwenk, F. W. 1987b. Nitrogen source utilization by chlorate-resistant isolates and chlorate-sensitive isolates of Macrophomina phaseolina. Tans. Br. Mycol. Soc. 88: 47–52. Pearson, C. A. S., Schwenk, F. W., Crowe, F. J. and Kelly, K. 1984. Colonization of soybean roots by Macrophomina phaseolina. Plant Dis. 68: 1086–1088. Penning de Vries, F. W. T. and Djitèye, M. A. (Eds). 1982. La production des pâturages sahéliens. Une étude des sols, des végétations et de l'exploitation de cette ressource naturelle. Agric. Res. Rep. 918, PUDOC, Wageningen. 523 p. Pullman, G. S., DeVay, J. E., Garber, R. H. and Weinhold, A. R. 1981. Soil solarization: Effects on Verticillium dahliae, Pythium, spp. Rhizoctonia solani, and Thielaviopsis basicola. Phytopathology 71: 954–959. Purkayastha, S., Kaur, B., Dilbahi, N. and Chaudhury, A. 2006. Characterization of Macrophomina phaseolina, the charcoal rot pathogen of cluster bean, using conventional techniques and PCR-based molecular markers. Plant Pathol. 55: 106–116. Purkayastha, S., Kaur, B., Dilbaghi, N. and Chaudhury, A. 2004. Cultural and pathogenic variation in the charcoal rot pathogen from clusterbean. Ann. Agri. Biol. Res. 9: 217–221. Ramirez-Villapudua, J. and Munnecke, D. E. 1987. Control of cabbage yellow (Fusarium oxysporum f. sp. conglutinans) by solar heating of field soil

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amended with dry cabbage residues. Plant Dis. 71 : 217–221. Reyes-Franco, M. C., Hernández-Delgado, S., Beas-Fernández, R., Medina,- Fernández, M., Simpson, J. and Mayek-Pérez, N. 2006. Pathogenic variability within Macrophomina phaseolina from Mexio and other countries. J. Phytopathol. 154: 447–453. Rose, E., Kabore, V. and Guenat, C. 1993. Le Zaï: fonctionnement, limites et amélioration d’une pratique traditionnelle africaine de réhabilitation de la végétation et de restauration de la productivité des terres dégradées de la région soudano-sahélienne. Cahiers ORSTOM série Pédologie 28: 159–174. Saxena, J. and Mathela, C. S. 1997. Antifungal activity of new compounds from Nepata leucophylla and Nepata clarkei. J. Appl. Env. Microbiol. 62: 702–704. Sharma, R. K., Aggarwal, R. K. and Lodha, S. 1995. Population changes of Macrophomina phaseolina and Fusarium oxysporum f. sp. cumini in oil-cake and crop residue-amended sandy soils. Appl. Soil Ecol. 2: 281–284. Sheikh, A. and Ghaffar, A. 1979. Relation of sclerotial inoculum density and soil moisture to infection of field crops by Macrophomina phaseolina. Pak. J. Bot. 11: 185–189. Short, G. E. and Wyllie, T. D. 1978. Inoculum potential of Macrophomina phaseolina. Phytopathology 68: 742–746. Short, G. E., Wyllie, T. D. and Ammon, V. D. 1978. Quantitave enumeration of Macrophomina phaseolina in soybean tissues. Phytopathology 68: 736–741. Short, G. E., Wyllie, T. D. and Bristow, P. R. 1980. Survival of Macrophomina phaseolina in soil and residue of soybean. Phytopathology 70: 13–17. Siddiqui, Z. A. and Mahmood, I. 1993. Biological control of Meloidogyne incognita race 3 and Macrophomina phaseolina by Paecilomyces lilacinus and Bacillus subtilis alone and in combination in chickpea. Fund. Appl. Nematol. 16: 215– 218. Sinclair, J. B. and Backman, P. A., eds. 1989. Compendium of soybean diseases. 3rd ed. American Phytopathological Society, St. Paul, MN. 106 p. Singh, B. B. 1994. Breeding suitable cowpea varieties for West and Central African savanna. Pp 77-85 in Progress in Food Grain Research and Production in Semiarid Africa, Menyonga, J. M., Bezuneh, T. B. Yayock, J. Y. and Soumana, I. eds. OAU/STRC-SAFGRAD, Ouagadougou, Burkina Faso. Singh, B. B. and Sharma, B. 1996. Restructuring cowpea for higher yield. Indian J. Genetics 56: 389–405. Singh, S. and Lodha, S. 1986. Varietal resistance of cowpea to Macrophomina phaseolina (Tass) Goid. causing dry root rot and its control. Indian J. Agric. Sci. 58: 552–555.

21 Chapter 1

Smith, G. S. and Carvil, O. N. 1997. Field screening of commercial and experimental soybean cultivars for their reaction to Macrophomina phaseolina. Plant Dis. 81: 363–368. Sohi, H. S. and Rawal, R. D. 1983. Field resistance of cowpea varieties to anthracnose (Colletotrichum lindemuthianum) and stem blight (Macrophomina phaseolina) diseases. Indian J. Mycol. Plant Pathol. 13: 58–60. Songa, W. and Hillocks, R. J. 1996. Legume hosts of Macrophomina phaseolina in Kenya and effect of crop species on soil inoculum level. J. Phythopathol. 144: 387–391. Srivastava, A. K. Arora, D. K., Gupta, S., Pandey, R. R. and Lee, M. 1996. Diversity of potential microbial parasites colonizing sclerotia of Macrophomina phaseolina in soil. Biol. Fertil. Soils 22: 136–140. Su, G., Suh, S. O., Schneider, R. W. and Russin, J. S. 2001. Host specialisation in the charcoal rot fungus, Macrophomina phaseolina. Phytopathology 91: 120–126. Tarawali, S. A., Singh, B. B., Peters, M. and Blade, S. F. 1997. Cowpea haulms as fodder. Pp 313-325 in Advances in cowpea research, Singh, B. B., Mohan Raj, D. R., Dashiell, K. E. and Jackai, L. E. N. eds. Copublication of the International Institute of Tropical Agriculture (IITA) and Japan International Research Center for Agricultural Sciences (JIRCAS). IITA, Ibadan, Nigeria. Usmani, S. M. H. and Ghaffar, A. 1982. Polyethylene mulching of soil to reduce viability of sclerotia of Sclerotium oryzae. Soil. Biochem. 14: 203–207. Villeneuve, F. 2000. Matière Organique et production intégrée. Pp 41-51 in Bulletin de Liaison numéro 17 FAO/GCP/RAF ed. Dakar. Voland, R. P. and Epstein, A. H. 1984. Development of suppressiveness to diseases caused by Rhizoctonia solani in soils amended with composted and non composted manure. Plant Dis. 78: 46–466. Watanabe, T., Smith, R. S. and Snyder, Jr. and W. C. 1970. Population of Macrophomina phaseoli in soil as affected by fumigation and cropping. Phytopathology 60: 1717–1719. Wrather, J., A., Anderson, T, R., Arsyad, D. M., Gai, J., Ploper, L. D., Porta-Puglia, A., Ram, H. H. and Yorinori, J. T. 1997. Soybean disease loss estimates for the top 10 soybean producing countries in 1994. Plant Dis. 81: 107–110. Wrather, J., A., Anderson, T, R., Arsyad, D. M., Tan, Y., Ploper, L. D., Porta-Puglia, A., Ram, H. H. and Yorinori, J. T. 2001. Soybean disease loss estimates for the top 10 soybean producing countries in 1998. Can. J. Plant Pathol. 23: 115–121. Wyllie, T. D. and Cavert, O. H. 1969. Effect of flower removal and pod set on the formation of sclerotia and infection of Glycine max by Macrophomina phaseoli. Phytopathology 59: 1243–1245.

22 General introduction

Young, D, J. and Alcorn, S. M. 1984. Latent infection of Euphorbia lathyris and weeds by Macrophomina phaseolina and propagule populations in Arizona field soil. Plant Dis. 68: 587–589. Zahid, M. I., Gurr, G. M., Nikandrow, A., Hodda, M., Fulkerson, W. J. and Nicol, H. I. 2002. Effects of root- and stolon-infecting fungi on root-colonizing nematodes of white clover. Plant Pathol. 51: 242–250. Zazzerni, A. and Tosi, L. 1989. Chlorate sensitivity of Sclerotium bataticola isolates from different hosts. Phytopathol. Z. 126: 219–224.

CHAPTER 2

Physiological, genetic, and pathogenic variability in Macrophomina phaseolina, the causal agent of charcoal rot

M. Ndiaye1, A. J. Termorshuizen2, A. H. C. van Bruggen2, J. Z. Groenewald3 and P. W. Crous3

Macrophomina phaseolina causes heavy yield loss to cowpea (Vigna unguiculata) grown in different cropping systems in the Sahel region of Africa. It was not known if cropping systems could influence physiological, genetic and pathogenic characteristics of M. phaseolina isolates. This study therefore aimed to analyze the population structure of M. phaseolina associated with three cropping systems in the Sahel. Seventy-five isolates of M. phaseolina were collected from soil, as well as cowpea and millet (Pennisetum glaucum) plants grown in three different Sahelian cropping systems, viz. continuous cowpea, cowpea in rotation with millet, and cowpea intercropped with millet. In vitro cultural growth of isolates was determined at six temperatures, and a subset of isolates was subjected to sequence analysis of the ITS (internal transcribed spacer) region of the nuclear ribosomal DNA gene. Pathogenicity of 63 isolates was determined on three cowpea varieties, and 20 isolates were also tested on fonio (Digitaria exilis), millet, maize (Zea mays), sorghum (Sorghum vulgare) and cowpea cv. Mouride. All M. phaseolina isolates formed microsclerotia at temperatures ranging from 25 to 40°C, though microsclerotial formation was found to vary among isolates. The optimal mycelial growth temperature on PDA was 34–37°C for most isolates. Isolate origin significantly affected the mycelial growth rate at 33°C, pathogenicity on cowpea cv. Mouride and on cereals, and the genotypic structure of the fungal population. The molecular analysis distinguished four genotypes from the field isolates collected in this study. An additional five genotypes were observed among cultures obtained from the CBS Fungal Biodiversity culture collection. Isolates collected from Senegal were distributed among three, and isolates from Niger in two genotypes, respectively. The dominant genotype was common between the two collection localities.

1 AGRHYMET/DFR BP. 12625 Niamey, Niger; email: [email protected] 2 Biological Farming Systems Group, Wageningen University, Marijkeweg 22, 6709 PG Wageningen, The Netherlands; email: [email protected] and [email protected] 3 Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre P.O. Box 85167, 3508 AD, Utrecht, The Netherlands; email: [email protected] and [email protected]

25 Chapter 2

Isolates originating from fields with a cowpea/millet rotation had a faster growth rate than isolates collected from other cropping systems. Isolates originating from continuous cowpea systems were more virulent on cv. Mouride, but colonized maize significantly less than isolates collected from fields of cowpea intercropped with millet or cowpea in rotation with millet. However, pathogenicity tests with 20 isolates of M. phaseolina on four cereal species indicated a similar host range. Fonio appeared to be a nonhost of the fungus, and millet, maize, and sorghum were susceptible to all isolates. In conclusion, isolates of M. phaseolina differed to a limited extent in their temperature requirements for growth, genetic make-up, and pathogenicity to cereal crops. The grouping according to physiological and genetic traits did not coincide with that based on pathogenicity. However, for the first time, we showed some specialization in pathogenicity to cereal crops (except fonio) for isolates obtained from fields grown to millet besides cowpea.

Introduction Macrophomina phaseolina (Tassi) Goid. is a soil- and seedborne polyphagous pathogen that causes charcoal rot and various rots and blights of more than 500 crop species (Sinclair and Backman, 1989; Dhingra and Sinclair, 1977). In the Sahelian zone of West Africa (including Burkina Faso, Niger and Senegal), charcoal rot causes on average a yield loss of 10%, which is equivalent with 30,000 tons cowpea with an estimated value of $ 146 millions only for Niger and Senegal. Although its telemorph is unknown, M. phaseolina is classified in the Botryosphaeriaceae according to recent phylogenetic data (Crous et al., 2006). The asexual structures formed by the fungus are pycnidia and microsclerotia. The black, 0.1–1 mm sized microsclerotia are formed in infected host tissues and constitute the primary inoculum source of the pathogen. They can survive up to 15 years depending on environmental conditions, and whether or not the sclerotia are associated with host residues (Cook et al., 1973; Papavizas, 1977; Short et al., 1980). Secondary dispersal by pycnidiospores is host- and isolate-dependent (Ali and Dennis, 1992). Many studies have been devoted to the morphological, physiological, and pathogenic variability of M. phaseolina (Mayek-Pérez et al., 2001; Reyes-Franco et al., 2006; Jana et al., 2005; Purkayastha et al., 2006). Manici et al. (1995) characterized 64 sunflower isolates of M. phaseolina collected from four different climatic zones in Italy. There was practically no difference between isolates in optimum temperature (30–35oC) for in vitro growth, and their pathogenicity on a range of hosts was similar. Some limited variation in chlorate sensitivity was noticed although the majority (95%) was chlorate tolerant. Contrary to host origin, mycelial

26 Physiological, genetic, and pathogenetic variability growth rate and sclerotial production in vitro predicted pathogenicity on cluster bean seedlings (Purkayastha et al., 2004). Isolates of M. phaseolina collected from field peas were differentiated into 15 pathotypes according to their pathogenicity. None of the tested pea varieties were immune, but adequate sources of resistance were identified against all isolates (Ali and Dennis, 1992). Mihail and Taylor (1995) reported that isolates of M. phaseolina collected from members of the Poaceae were less virulent on sunflower cv. Mammoth and Euphorbia lathyris than those collected from Asteraceae, Euphorbiaceae or Fabaceae. Among 96 isolates collected from Mexico and other countries, nine (from Japan, Italy, Brazil, USA, and Australia) were not pathogenic on bean (Reyes-Franco et al., 2006). Recently, investigations on the molecular analysis of isolates of M. phaseolina have contributed to the better understanding of the relationship between the populations of the fungus and their origin (host or geographic location). Among 84 isolates of M. phaseolina collected from different hosts and geographical origin, Mayek-Pérez et al. (2001) distinguished 84 amplified fragment length polymorphism (AFLP) genotypes, but only 43 pathotypes on the basis of their reaction to 12 differential cultivars of common bean. Cluster analysis of a subset of the isolates obtained from common bean or from the state of Tamaulipas (Mexico), indicated a tendency for isolates to form groups related to geographical and host origin. Molecular analysis of the genomic DNA of 70 isolates of M. phaseolina using RAPD (random amplified polymorphic DNA) clearly distinguished the isolates on the basis of chlorate phenotype and host origin (Purkayasta, et al., 2006). Furthermore, Jana et al. (2003) studying the genetic variation in 43 isolates of M. phaseolina and 22 isolates of Fusarium species from India, North America and other locations using RAPD, found that one primer OPA-13 was able to distinguish between M. phaseolina and a Fusarium sp. at the level of isolates, formae-speciales and races.

This work aimed to study the population structure of M. phaseolina associated with three cultivation systems of cowpea, and to characterize the isolates with respect to in vitro growth rate, genotypic variability, and pathogenicity to cowpea and other cereal crops.

Material and methods

Field survey and collection of fungal isolates During 1990–1991, collections of Macrophomina phaseolina were made from the major cowpea (Vigna unguiculata (L.) Walp.) production areas of Senegal (regions of Diourbel, Louga, and Thies) and in 1996–1997 in those of Niger (districts Niamey,

27 Chapter 2

Dosso, and Gaya). All areas had an annual rainfall of 350–500 mm except Gaya (500– 855 mm) and Louga (200–350 mm). There were two types of cowpea production systems in Niger: cowpea monoculture and cowpea/millet intercropping, while in Senegal the cropping systems were cowpea/millet rotation and cowpea monoculture. In Senegal, soil cores 0–15 cm deep were collected with an auger at the beginning of the cropping season before planting. In Niger, stems and roots of symptomatic plants were sampled from farmers’ fields and experimental plots. Depending on the number of diseased foci in a trial, 3–5 plants were sampled per field. Whenever possible, the cropping history of the fields was recorded. All samples were deposited at the CILSS/AGRHYMET Phytopathology Laboratory in Niamey before processing.

Isolation and identification of Macrophomina phaseolina From soil. A soil-assay technique based on Alabouvette (1976) was used. Sampled soil was dried in an oven at 37°C, crushed through a 1-mm sieve, mixed thoroughly, and subsamples of 5 g were taken with a spoon. Each subsample of 5 g was treated with 0.525% NaOCl for 10 min. If isolation was unsuccessful due to contamination an additional subsample was treated with a 0.525% NaOCl for 20 min. The soil-NaOCl mixture was washed with distilled water through two sieves of 180 and 45 µm. The residue retained on the 45-µm sieve was incorporated into 100 ml of a semi-selective medium (SS medium) for M. phaseolina that was prepared as follows. To potato- dextrose agar (PDA; 39 g l-1) maintained at 55°C in a water bath the following ingredients were added: 1.5 ml 0.525% NaOCl, 1 ml 0.5% choramphenicol dissolved in 95% alcohol, and 10 ml 2.25% quintozene (PCNB). This medium was poured in 10 Petri dishes and incubated at 30°C for 5–8 d. From host tissue. Uprooted plants were rinsed well under running tap water, and blotted dry with a sterile paper towel. Separate subsamples of roots and stems of each plant were treated by immersion in 0.5% NaOCl for 10 min to eliminate secondary invaders followed by rinsing for 30 min in sterile distilled water. Tissues excised from dying rootlets, necrotic taproots and stems were cut into 2–3 mm long fragments, and 15 of these were plated on three Petri dishes containing the above- described SS-medium. Plated Petri dishes were incubated at 30°C for 5–8 d. Colonies of M. phaseolina appeared as a ring of fluffy white mycelium surrounding a central area with black microsclerotia. Each colony was observed under the microscope for the presence of microsclerotia and then propagated further onto PDA, and stored at 5°C. Isolates were also preserved after mixing pure cultures with sterile dry soil (1 plate 20 g-1 soil) and storing at 5°C. Among the studied isolates, 31 originated from Niger and 44 from Senegal. Thirty-nine percent of all isolates were collected from cowpea as sole crop, 29% from cowpea/millet intercrop, and 32% from

28 Physiological, genetic, and pathogenetic variability cowpea/millet rotation (Table 1). Additional cultures of M. phaseolina (= Tiarosporella phaseolina (Tassi) van der Aa) were obtained for molecular comparison from the culture collection of the CBS Fungal Biodiversity Centre (CBS) in Utrecht, The Netherlands, and the culture collection of Pedro Crous (CPC) housed at CBS.

Table 1. Origin, lab number, and codes of Macrophomina phaseolina field isolates used in this study.

Cropping system Origin Country Village Code number Lab code

Cowpea continuous culture Cowpea Niger Bara 2 BaraTN2 Cowpea continuous culture Cowpea Niger Bara 14 Bara TN7 Cowpea continuous culture Cowpea Niger Bara 21 Bara TN8 Cowpea continuous culture Cowpea Niger Bara 52 Bara RN1 Cowpea continuous culture Cowpea Niger Bara 53 Bara TN4 Cowpea continuous culture Cowpea Niger Bara 54 Bara TN5 Cowpea continuous culture Cowpea Niger Bara 55 Bara TN9 Cowpea continuous culture Cowpea Niger CRA (Niamey) 26 PiTN2 Cowpea continuous culture Cowpea Niger CRA (Niamey) 42 CATN5 Cowpea continuous culture Soil Senegal Bambey 60 Bam1 Cowpea continuous culture Soil Senegal Bambey 61 Bam2 Cowpea continuous culture Soil Senegal Bambey 62 Bam3 Cowpea continuous culture Soil Senegal Bambey 63 Bam4 Cowpea continuous culture Soil Senegal Bambey 66 Bam7 Cowpea continuous culture Soil Senegal Bambey 67 Bam8 Cowpea continuous culture Soil Senegal Bambey 68 Bam9 Cowpea continuous culture Soil Senegal Bambey 69 Bam10 Cowpea continuous culture Soil Senegal Bambey 70 Bam11 Cowpea continuous culture Soil Senegal Bambey 71 Bam12 Cowpea continuous culture Soil Senegal Bambey 72 Bam13 Cowpea continuous culture Soil Senegal Bambey 73 Bam14 Cowpea continuous culture Soil Senegal Bambey 75 Bam16 Cowpea continuous culture Soil Senegal Bambey 76 Bam17 Cowpea continuous culture Soil Senegal Bambey 77 Bam18 Cowpea continuous culture Soil Senegal Bambey 78 Bam19 Cowpea continuous culture Soil Senegal Bambey 80 Bam21 Cowpea continuous culture Soil Senegal Bambey 81 Bam22

29 Chapter 2

Cowpea continuous culture Soil Senegal Bambey 82 Bam23 Cowpea continuous culture Soil Senegal Bambey 83 Bam24 Cowpea/millet intercrop Cowpea Niger Gabougoura 10 GGTMN2 Cowpea/millet intercrop Cowpea Niger Gabougoura 11 GGRMN6 Cowpea/millet intercrop Cowpea Niger Gabougoura 12 GGTMN3 Cowpea/millet intercrop Cowpea Niger Gabougoura 13 GGTMN4 Cowpea/millet intercrop Cowpea Niger Bara 23 Bara RMN2 Cowpea/millet intercrop Cowpea Niger Bara 24 Bara RMN3 Cowpea/millet intercrop Cowpea Niger Bengou 28 BeRMN1 Cowpea/millet intercrop Cowpea Niger Bengou 29 BeRMN2 Cowpea/millet intercrop Cowpea Niger Bengou 30 BeRMN3 Cowpea/millet intercrop Cowpea Niger CRA (Niamey) 38 CATMN1 Cowpea/millet intercrop Cowpea Niger CRA (Niamey) 39 CATMN2 Cowpea/millet intercrop Cowpea Niger CRA (Niamey) 40 CATMN3 Cowpea/millet intercrop Cowpea Niger CRA (Niamey) 41 CATMN4 Cowpea/millet intercrop Cowpea Niger CRA (Niamey) 43 CATMN5 Cowpea/millet intercrop Cowpea Niger CRA (Niamey) 44 CATMN6 Cowpea/millet intercrop Cowpea Niger Gabougoura 58 GGRMN2 Cowpea/millet intercrop Millet Niger Gabougoura GM1 GGRM1 Cowpea/millet intercrop Millet Niger Gabougoura GM2 GGRM2 Cowpea/millet intercrop Millet Niger Gabougoura GM3 GGRM3 Cowpea/millet intercrop Millet Niger Gabougoura GM5 GGRM5 Cowpea/millet intercrop Millet Niger Gabougoura GM6 GGRM6 Cowpea/millet intercrop Millet Niger Gabougoura GM7 GGRM7 Cowpea/millet rotation Soil Senegal Kebemer 86 Kebe2 Cowpea/millet rotation Soil Senegal Kebemer 87 Kebe3 Cowpea/millet rotation Soil Senegal Kebemer 88 Kebe4 Cowpea/millet rotation Soil Senegal Kebemer 89 Kebe5 Cowpea/millet rotation Soil Senegal Kebemer 90 Kebe6 Cowpea/millet rotation Soil Senegal Kebemer 91 Kebe7 Cowpea/millet rotation Soil Senegal Kebemer 92 Kebe8 Cowpea/millet rotation Soil Senegal Kebemer 93 Kebe9 Cowpea/millet rotation Soil Senegal Kebemer 94 Kebe10 Cowpea/millet rotation Soil Senegal Kebemer 96 Kebe12 Cowpea/millet rotation Soil Senegal Kebemer 97 Kebe13 Cowpea/millet rotation Soil Senegal Kebemer 98 Kebe14 Cowpea/millet rotation Soil Senegal Kebemer 100 Kebe1 Cowpea/millet rotation Soil Senegal Kebemer 101 Kebe16

30 Physiological, genetic, and pathogenetic variability

Cowpea/millet rotation Soil Senegal Kebemer 102 Kebe17 Cowpea/millet rotation Soil Senegal Kebemer 103 Kebe18 Cowpea/millet rotation Soil Senegal Kebemer 104 Kebe19 Cowpea/millet rotation Soil Senegal Kebemer 105 Kebe20 Cowpea/millet rotation Soil Senegal Kebemer 107 Kebe22 Cowpea/millet rotation Soil Senegal Kebemer 108 Kebe23 Cowpea/millet rotation Soil Senegal Kebemer 109 Kebe24 Cowpea/millet rotation Soil Senegal Kebemer 110 Kebe25 Cowpea/millet rotation Soil Senegal Kebemer 111 Kebe26

Effects of temperature on growth rate of isolates of Macrophomina phaseolina Cultural growth rate of all isolates was compared on PDA plates incubated at 25, 27, 30, 33, 35, and 37°C. Culture disks, 4 mm diameter, cut from the edge of a 4-day-old PDA culture grown at 30°C, and transferred to a 9-cm Petri dish containing 10 ml PDA. Plates were incubated at the six different temperatures. Each treatment was replicated three times. Colony diameter was recorded daily, starting 24 h after plating and continued for 3–4 d.

DNA isolation, amplification and phylogeny Fungal colonies were established on PDA plates, and genomic DNA was isolated following the protocol of Lee and Taylor (1990). The primers V9G (Hoog and Gerrits van den Ende, 1998) and ITS4 (White et al., 1990) were used to amplify part (ITS) of the nuclear rDNA operon spanning the 3’ end of the 18S rDNA gene (SSU), the first internal transcribed spacer (ITS1), the 5.8S rDNA gene, the second ITS region and the 5’ end of the 28S rDNA gene (LSU). The PCR conditions, sequence alignment and subsequent phylogenetic analysis followed the methods of Crous et al. (2004). Genotypes were assigned by visually scoring the differences in the sequences resulting in the obtained phylogenetic tree. To ensure high quality sequences, both strands of the PCR products were sequenced and the genotypic differences were confirmed by manual inspection of the raw sequence data.

Pathogenicity tests Inoculum preparation. Inoculum of the isolates for the bioassays was prepared as follows. Millet cv. HKP was soaked during 24 h in distilled water, excess water was eliminated, 50 g d.w. of the soaked grains were placed in 250 ml flasks and subsequently sterilized at 121°C for 30 min. After cooling, the grains in the flask were

31 Chapter 2 inoculated with an isolate of M. phaseolina by placing six 5-mm discs cut out from a 3-d-old fungal culture growing on PDA. The inoculated flasks were then incubated during 15 d at 30°C, dried at 37–40°C in an oven for 5 d, placed in plastic bags, sealed, and preserved at 4°C until use. Sterilized soil was infested with the millet inoculum at a rate of 5% (w/w; Mayek-Peréz et al., 2001). Cowpea experiments. The pathogenicity of 63 isolates was studied on three cowpea cultivars in the screenhouse in a split plot design with cowpea varieties as main plots and the isolates as subplots. Treatments were repeated three times. While in sorghum resistance to M. phaseolina is related to genotypes exhibiting delayed leaf senescence (Ducan, 1984; Diourte et al., 1995), the existence of this relation was not known for cowpea. Also, in an additional experiment, the reaction of delayed leaf senescence typical for cowpea cv. Mouride to the 20 isolates was tested in a completely randomized block design. The crop characteristics and varieties used are given in Table 2. Before planting, an indigenous Rhizobium sp. strain ISRA 311, kindly provided by dr. Mamadou Gueye, MIRCEN ISRA/IRD (Senegal), was used to coat surface- disinfested (2.5% NaOCl for 5 min) cowpea seeds. The bacteria were grown on tryptic soy broth (TSB) medium at 28°C for 48 h. Bacterial colonies were aseptically collected in 60 ml sterilized distilled water (SDW). The suspension was then centrifuged for 15 min at 5000 rpm min-1 in a SIGMA 3–12 laboratory centrifuge. The supernatant was discarded and the bacterial pellet adjusted to a cell density of 109 colony forming units (cfu) ml-1 with SDW. Cowpea seeds were then dipped in the bacterial suspension for 15 min. The coated seeds (3 seeds per pot) were sown in 3 1-L pots containing inoculum (100 microsclerotia g-1 soil) of one of the isolates of M. phaseolina. Pots without inoculum served as controls. The pots were then placed on a tray and incubated in a screenhouse at 30 ± 5°C and 70 ± 20% RH recorded with a thermohydrograph. The number of diseased and dead plants was recorded weekly. The plants were uprooted 45 days after sowing. The root systems were washed with tap water, blotted dry with a paper towel and the roots and stems were air-dried in an oven at 37°C. Subsequently the dried tissues were milled, 150 mg of the powder was mixed with 100 ml of the SS-medium and poured in 10 petri dishes. The plates were incubated at 30°C for 10 d and the number of germinated microsclerotia counted. The disease intensity and area under the disease progress curve (AUDPC) were also calculated. Cereal experiment. Twenty isolates of M. phaseolina were used to inoculate maize (Zea mays), pearl millet (Pennisetum glaucum), sorghum (Sorghum bicolor), and fonio (Digitaria exilis) plants under similar conditions in a randomized complete

32 Physiological, genetic, and pathogenetic variability block design with three replications. Among these isolates, there were 12 from Senegal and eight from Niger. Seven isolates were collected from continuous cowpea fields, seven from cowpea/millet intercrop fields, and six from cowpea/millet rotation fields. Nine, three, five, and 10 seeds of millet, maize, sorghum, and fonio, respectively, were sown per pot containing infested and non-infested soils. Pots were placed in a screenhouse completely randomized block design with three replications, and watered as needed. Ten days after sowing the germination rate of crops was recorded and weekly the number of dead plants counted. At 45 days after sowing, the plants were uprooted and the roots and stems analyzed for the number of sclerotia as describe as above.

Table 2. Characterization of crop species.

Crop Variety Breeding number Cycle sowing to Adaptation zone maturity (days) Cowpea Dan bobo KVx 30-309 6G 70–75 300–800 mm Dan louma TN5–78 70–75 300–800 mm TVx 3236 TVx 3236 60 350–600 mm Mouride IS86-275 66 250–600 mm Millet HKP - 90–95 350–500 mm Maize P3 Kolo - 90 500–600 mm Sorghum IRAT 204 IRAT 204 90 500–700 mm Fonio Local variety - 90 500–700 mm

Data analysis Data were analyzed with Genstat® for Windows 8th Edition (IACR-Rothamsted, Harpenden, Hertfordshire, UK). The data were subjected to analysis of variance. Treatment means were separated by the Duncan’s Multiple Range Test (DMRT). Numbers of microsclerotia in plant tissues were analyzed after log transformation if necessary. To compare isolates with regard to their growth rate at different temperatures 3 d after plating, an analysis of variance for a RCDB was run on collected data. The DMRT separated treatment means. Pathogenicity comparison of M. phaseolina isolates and evaluation of host species were performed in factorial experiments. Each plastic pot was considered to be an experimental unit. In order to assess any relationship between the isolates, principal component analysis (PCA) was carried out on quantitative variables used for isolate characterization: average growth rate over six temperatures [grmean], optimal growth temperature [opt], diameter of

33 Chapter 2 fungal colony at 37°C after 3 d incubation [diam], diameter of sclerotial zone at 37°C after 3 d incubation [scl37], density of sclerotial zone [scldens], genotype group [ITS], Area Under Disease Progress Curve [AUDPC], and density of sclerotia in millet [sclmil], maize [sclmaize] and sorghum [sclsor]. For PCA the multivariate statistical program CANOCO version 4.5 (ter Braak and Šmilauer, 2002) was used.

Results

Growth at different temperatures The optimal temperature for in vitro growth was 35–37°C for 92% of isolates. At 25 and 27°C, the mean growth rate was about 25% slower than at optimal temperatures (Fig. 1). The differences in growth at the various temperatures were significant (P < 0.01) for all isolates. There was a very sharp decline in fungal growth above 37oC. In general there was no effect of isolate on colony diameter except for the group of isolates originating from cowpea/millet rotation, which grew significantly faster (8%) at 33°C than the other isolates (P < 0.05). Other in vitro growth characteristics also varied little among isolates, such as the size of the microsclerotial zone and culture appearance (fluffy, aerial, or submerged mycelium).

ITS sequence analysis The ITS alignment consisted of 88 taxa including the two outgroups and 506 characters (including alignment gaps) were included in the analyses. Of these characters, 56 were parsimony-informative, 52 were variable and parsimony- uninformative, and 398 were constant. Parsimony analysis with gaps treated as new states yielded two equally most parsimonious trees, one of which is shown in Fig. 2. Nine genotypes were identified in the phylogram and confirmed by visual inspection of the sequence alignment. The fixed nucleotide states and locations responsible for the genotypes are listed in Table 3. The majority of isolates, including all but one of the Niger isolates, belong to genotype 1. Isolates from Senegal were represented by three genotypes namely 1, 5 and 9. Genotype 4 is represented by a single strain isolated from cowpea in Niger and differs with one nucleotide from the rest of the genotype 1 isolates. Isolates of genotype 9 produced higher densities of microsclerotia (P = 0.03) on cowpea cvs. KVx, TN5-78 and TVx than isolates of genotypes 1 and 5 (Fig. 3). Genotypes 2, 3, and 6–8 are represented by strains isolated from a variety of hosts and countries. The rest of the CBS strains belong to genotype 1. Genotypes 2–4 and 8 can be derived from genotype 1 by single nucleotide changes, whereas genotypes 5, 6 and 9 are derived by insertion or deletion ("indel") events (Table 3).

34 Physiological, genetic, and pathogenetic variability

Fig. 1. Colony diameter (cm) of Macrophomina phaseolina isolates grown at various temperatures for 72 h on potato dextrose agar. The isolates originated from three cropping systems: CCC: continuous cowpea cultivation, CMI: cowpea/millet intercrop or CMR: cowpea/millet in rotation.

Genotype 7 is derived from genotype 1 by both a single nucleotide change and an indel event or from genotype 6 by a single nucleotide change (Table 3).

Density of microsclerotia in cowpea root and stem tissues and area-under-disease- progress-curve (AUDPC) On average, M. phaseolina produced 869 ± 96 microsclerotia g-1 d.w. of cowpea tissue (roots and stems together). The production depended of cowpea cultivar: 2.6x less microsclerotia were present in cv. TVx than in the other cultivars (P < 0.01) (Fig. 4). However, there was no effect of isolate or its origin on the number of sclerotia in cowpea tissue. In contrast the origin of the isolates significantly affected the AUDPC in cowpea cv. Mouride (Fig. 5). Isolates collected from cowpea continuous cultivation induced more disease than isolates originated from cowpea and millet intercropping or from cowpea in rotation with millet.

35 Chapter 2

Fig. 2. One of two equally most parsimonious trees obtained from a heuristic search with simple taxon additions of the ITS sequence alignment of isolates of Macrophomina phaseolina. The scale bar shows a single change, and bootstrap support values from 1000 replicates are shown at the nodes. All branches were present in the strict consensus tree. The tree was rooted to Tiarosporella madreeya CBS 532.76 and Botryosphaeria rhodina AY236952.

36 Physiological, genetic, and pathogenetic variability

Table 3. The locations and nucleotide states specific to each genotype observed in the ITS1 and ITS2 regions of the sequenced strains. Locations correspond to the position of the character(s) in the sequence of Macrophomina phaseolina strain P207 (GenBank Accession number AF132795). The position of extra characters (indel events) is indicated in the table by the separation of the two flanking character positions with a slash symbol. Characters identical to those in GenBank accession number AF132795 are indicated with a double quotation symbol.

Locations and identities of fixed nucleotide states 15/16 24/25 78/79 105 142 156 160/161 175 375/376 478 AF132795 G C T T T Genotype 1 " " " " " Genotype 2 T " " " " Genotype 3 " " " " C Genotype 4 " " " C " Genotype 5 G C T " " " " " Genotype 6 " " " A " " Genotype 7 " " G A " " Genotype 8 " T " " " Genotype 9 " " " " GTCC "

Fig. 3. Microsclerotial density in cowpea (cvs. KVx, TN5-78 and TVx) as function of ITS genotype group 45 days after sowing (number of isolates tested for genotypes 1, 5, and 9: 45, 3 and 10 respectively). Letters above bars indicate significant differences (P < 0.05).

37 Chapter 2

Fig. 4. Effect of cowpea variety on the number of microsclerotia of M. phaseolina in cowpea tissue 45 days after sowing in infested soil in pots. Letters above bars indicate significant differences (P < 0.05).

Fig. 5. Effect of origin of isolates of M. phaseolina on the area-under-disease-progress curve (AUDPC) in cowpea cv. Mouride 45 days after sowing in infested soil in pots. CCC: continuous cowpea cultivation (7 isolates tested), CMI: cowpea/millet intercropping (7 isolates), CMR: cowpea/millet rotation (6 isolates). Vertical bars denote the standard error of means.

Pathogenicity on cereals All tested species except fonio were susceptible to M. phaseolina. Millet was significantly (P = 0.01) less infected (35 sclerotia g-1 d.w. of tissue) than sorghum and maize (119 and 165 sclerotia g-1 d.w. of tissue, respectively) (Fig. 6).

38 Physiological, genetic, and pathogenetic variability

With respect to their origin, isolates derived from continuously cultivated cowpea (CCC) formed significantly (P = 0.02) less microsclerotia on maize than isolates from the other cropping systems (CMI and CMR), and those originating from cowpea/millet intercropping (CMI) produced less microsclerotia on maize than those originating from cowpea/millet rotation (CMR) (Fig. 7).

Fig. 6. Density of microsclerotia of M. phaseolina in roots and stem tissues of millet, maize and sorghum 45 days after sowing in infested soil in pots. Each crop was inoculated with 20 isolates originated from CCC: continuous cowpea cultivation (7 isolates), CMI: cowpea/millet intercrop (7 isolates) and CMR: cowpea/millet rotation (6 isolates). Vertical bars denote the standard error of means.

Fig. 7. Density of microsclerotia of M. phaseolina g-1 d.w. of maize roots and stems as influenced by isolate origin. CCC: continuous cowpea cultivation (7 isolates tested); CMI: cowpea/millet intercrop (7 isolates); CMR: cowpea/millet rotation (6 isolates). Vertical bars denote the standard error of means.

39 Chapter 2

91 1.0 58 scl-maiz

׀ 100 ׀ ׀aflpits GM1 avg-diam scl-37C diam-37C GM3 55 60 opt-temp 41% 82 44 63 GM7 79 90 81 38 scl-mil 107 98 scl-dens audpc

78 scl-sorg

GM2 -1.0 -0.6 58% 1.0

Fig. 8. Principal component analysis (PCA) of 20 isolates of M. phaseolina based on AUDPC (Area-Under-the-Disease-Progress-Curve) on cowpea (audpc), number of sclerotia produced in millet, maize and sorghum plants g-1 d.w. (scl-mil, scl-maiz, and scl-sorg respectively), number of microsclerotia formed in vitro, the diameter and density of the microsclerotial zone at 37°C (scl-37C, diam-37C and scl-dens respectively), in vitro growth rate averaged over 6 temperatures (avg-diam), optimal growth temperature (opt-temp), and ITS genotype (its). Isolate numbers correspond with those mentioned in Table 1.

40 Physiological, genetic, and pathogenetic variability

All isolates tested were pathogenic; however, their virulence expressed as number of sclerotia g-1 of tissue appeared highly variable. The results are summarized using a Principal Components Analysis (PCA) (Fig. 8). The results show that many isolates have a quite limited variation with respect to the parameters estimated in this study, and that isolates are best separated by the density of microsclerotia formed on maize and sorghum (scl-maize and scl-sor respectively; Fig. 8). Four isolates (58, 91, 100 and GM1) were relatively high in production of microsclerotia on maize. These isolates were collected from cowpea/millet intercropping or cowpea/millet rotation sites, but not from cowpea continuous culture sites. Three isolates (78, 97, and GM2) produced a high number of microsclerotia on sorghum and two isolates 79, 81, produced more sclerotia in millet than in maize or sorghum. Isolates 38, 101, 98, and 107 produced relatively high microsclerotial densities in sorghum. There were 15 isolates that produced more than 100 microsclerotia g-1 tissue of cereals, 11 derived from cowpea millet intercropping or cowpea alternating with millet.

Discussion The most suitable temperature for M. phaseolina growth was 35–37°C (8.0–8.6 cm in 72 h). Growth was slower at 25 and 27°C (3–5 cm in 72 h), but colonies of all isolates expanded up to 9 cm (diameter of Petri dish) within 5 d after plating. At 40°C, growth was slight (up to 2 cm after 72 h) or absent. Studying in vitro growth of M. phaseolina isolates from sunflower grown in different climatic regions of Italy, Manici et al. (1995) noticed that some isolates from the south (Mediterranean climate) grew better at 35°C. Also, Adam (1986) found that the optimum growth temperature of isolates collected in different areas of Niger was mostly 35°C, while fewer isolates grew optimally at 25°C or 30°C. However, we were unable to link isolate characteristics to growth aptitude although there was some difference among the isolates: 64% of the isolates had an optimum growth temperature of 35°C, while only 8% had an optimum growth temperature of 30°C. Four genotypes could be recognized based on the ITS sequence data in the 57 isolates tested from Niger and Senegal with genotype 1 being the most common and genotypes 5 and 9 being restricted to Senegal. Mayek-Peréz et al. (2001) found that all 84 isolates comprising 43 pathotypes of M. phaseolina had a unique AFLP-analysed genotype indicating an absence of correlation between pathotypes and genotypes of isolates of M. phaseolina. Su et al. (2001) were unable to find variation in restriction and length polymorphisms of the ITS region among 45 isolates of M. phaseolina obtained from maize, cotton, sorghum and soybean. In contrast, we did observe some genotypic variation based on the same region of the genome. However, most of the genotypes can be derived from the most common genotype (genotype 1) by single

41 Chapter 2 mutation events. More genes should therefore be sequenced to confirm these genotypes. Our results indicate that genotype 9 was more virulent on cowpea than genotypes 1 and 5. However, the numbers of isolates in genotype 9 (10) and genotype 5 (3) were small and may not be representative for the general populations. No isolate was pathogenic on fonio, but all were so on cowpea, maize, sorghum, and millet. However, as expected there were great differences among these hosts, with millet, sorghum and maize having lower densities of microsclerotia than cowpea. Maize appeared to be a reasonably good host for M. phaseolina. These findings corroborate our field observations (where fonio and millet did not show any charcoal rot disease symptoms) and the results of Ndiaye et al. (Chapter 5). There was appreciable variation in pathogenicity among isolates of M. phaseolina with respect to host and cropping system origin. Mihail and Taylor (1995) and Mayek-Pérez et al. (2001) have also found high variability in the general population of M. phaseolina with respect to host reaction. The last authors identified 45 different pathotypes from a total of 84 isolates of M. phaseolina using 12 differential cultivars of Phaseolus vulgaris. In our study, isolates (GM1, GM2, GM3, GM5, GM6 and GM7) collected from millet roots were virulent on cowpea and cereals, which disagrees with the results reported by Manici et al. (1995) about the limited pathogenicity of isolates collected from host members of the Poaceae. In our study, isolates derived from cropping systems with continuous cowpea cultivation were more virulent with respect to cowpea than isolates collected from cowpea cropping systems where millet was cultivated either by intercropping or in rotation with cowpea. Isolates from fields continuously cropped with cowpea formed significantly fewer microsclerotia in maize tissue than isolates derived from cowpea in rotation with millet or cowpea/millet intercropping. This is in agreement with the results of Mayek-Pérez et al. (2001), who showed that the majority of isolates of M. phaseolina infecting the most resistant P. vulgaris cultivars were collected from P. vulgaris. In conclusion, both molecular characterization (ITS sequence data) and pathogenicity tests (more sclerotia in maize from cowpea/millet isolates than from continuous cowpea isolates) indicate that some specialization occurs in M. phaseolina. Increased pathogenicity in fields cultivated continuously to the same crop due to selection is a common phenomenon (Francl al., 1988; Lebreton et al., 2004). Halving of the AUDPC on cowpea cv. Mouride by isolates of cowpea + millet (both intercropped and rotated) compared to continuously cropped cowpea (Fig. 5) is probably due to selection of part of the population that can infect monocotyledonous hosts. This is confirmed by the higher standard deviations of the average AUDPC on cowpea for the isolates from the cowpea + millet cropping systems (CMI and CMR,

42 Physiological, genetic, and pathogenetic variability

Fig. 5; 6.2 and 6.0 respectively) compared to the continuous cowpea cropping system (CCC; 5.2). On the other hand, a greater proportion of millet-specialized strains of M. phaseolina in the cowpea + millet cropping systems resulted in more microsclerotia on maize than those produced by isolates from the continuous cowpea system (Fig. 7). In conclusion, there are two contradictory effects of mixed cropping with respect to M. phaseolina: cowpea + millet cropping systems invoke 1.6–2.6 × (Fig. 7) higher numbers of microsclerotia on maize, but 1.4–2.0 × lower disease severity levels (expressed as AUDPC, Fig. 5) in cowpea. Given that cowpea is a much more important crop than maize, we conclude that mixed cropping systems with cowpea and millet are preferred and that alternating cowpea with maize or sorghum should be avoided. Being a nonhost, fonio can be used to reduce soil inoculum levels where charcoal rot disease is a recurrent problem. Finally, more isolates of genotype 5 and 9 types should be characterized to know their distribution and host specificity in terms of pathogenicity for better management of charcoal rot in Sahelian cultivation systems.

References Adam, T. 1986. Contribution à la connaissance des maladies du niébé (Vigna unguiculata (L.) Walp.) au Niger avec mention spéciale au Macrophomina phaseolina (Tassi) Goïd. Université de Renne I. Thèse de doctorat. 117 p. Alabouvette, C. 1976. Recherches sur l'écologie des champignons parasites dans le sol. VIII. - Etude écologique de Macrophomina phaseolina grâce à une technique d'analyse sélective. Ann. Phytopathol. 8:147–157. Ali, S. M. and Dennis, J. 1992. Host range and physiologic specialisation of Macrophomina phaseolina isolated from field peas in South Australia. Aust. J. Exp. Agric. 32: 1121–1125. Cook, G. E., Boosalis, M. G., Dunkle, L. D. and Odvody, G. N. 1973. Survival of Macrophomina phaseoli in corn and sorghum stalk residue. Plant Dis. Rep. 57: 873–875. Crous, P. W., Groenewald, J. Z., Pongpanich, K., Himaman, W., Arzanlou, M. and Wingfield, M. J. 2004. Cryptic speciation and host specificity among Mycosphaerella spp. occurring on Australian Acacia species grown as exotics in the tropics. Studies Mycol 50: 457–469. Crous, P. W., Slippers, B., Wingfield, M. J., Rheeder, J., Marasas, W. F. O., Philips, A. J. L., Alves, A., Burgess, T., Barber, P. and Groenewald, J. 2006. Phylogenetic lineages in the Botryosphaeriaceae. Studies Mycol. 55: 235–253. Dhingra, O. D. and Sinclair, J. B. 1977. An annotated bibliography of M. phaseoli, 1905–1975. Universidade Federal, Visçosa, Brazil. 277 p. Diourte, M., Starr, J. L., Jeger, M. J., Stack, J. P. and Rosenow, D. T. 1995. Charcoal

43 Chapter 2

rot (Macrophomina phaseolina) resistance and the effects of stress on disease development in sorghum. Plant Pathol. 44: 196–202. Ducan, R. R. 1984. The association of plant senescence with root and stalk diseases in sorghum. Pp 99–110 in Mughogho, L. K. ed. Sorghum Root and Stalk Rots: A Critical Review. Pantacheru, India: International Crop Research Institute for the Semi-Arid Tropics. Francl, L. J., Wyllie, T. D. and Rosenbrock, S. M. 1988. Influence of crop rotation on population density of Macrophomina phaseolina in soil infested with Heterodera glycines. Plant Dis. 72: 760–764. Hoog, G. S. de and Gerrits van den Ende, A. H. G. 1998. Molecular diagnostics of clinical strains of filamentous Basidiomycetes. Mycoses 41: 183–189. Jana, T., Sharma, T. R., Prassad, R. D. and Arora. D. K. 2003. Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique. Microbiol. Res. 158: 249–257. Jana, T., Singh, N. K., Koundal, K. R. and Sharma, T. R. 2005. Genetic differentiation of charcoal rot pathogen, Macrophomina phaseolina, into specific groups using URP-PCR. Can. J. Microbiol. 51: 159–164. Lebreton, L., Lucas, P., Dugas, F., Guillerm, A. Y., Schoeny, A. and Sarniguet, A. 2004. Changes in population structure of the soilborne fungus Gaeumannomyces graminis var. tritici during continuous wheat cropping. Env. Microbiol. 6: 1174–1185. Lee, S. B. and Taylor, J. W. 1990. Isolation of DNA from fungal mycelia and single spores. In: PCR Protocols: a guide to methods and applications (eds. M.A. Innis, D.H. Gelfand, J. J. Sninisky and T. J. White). Academic Press, San Diego, USA: 282–287. Manici, L. M., Caputo, R. and Cerato, C. 1995. Temperature responses of isolates of Macrophomina phaseolina from different climatic regions of sunflower production in Italy. Plant Dis. 79: 834–838. Mayek-Pérez, N., López-Castañeda, C., Gonzáles-Chavira, M., Garcia-Espenosa, R., Acosta-Gallegos, J., Martinez de Vega, O. and Simpson, J. 2001. Variability of Mexican isolates of Macrophomina phaseolina based on pathogenesis and AFLP genotype. Physiol. Molec. Plant Pathol. 59: 257–264. Mihail, J. D. and Taylor, S. J. 1995. Interpreting variability among isolates of Macrophomina phaseolina in pathogenicity, pycnidium production, and chlorate utilization. Can. J. Bot. 73: 1596–1603. Olanya, O. M. and Campbell, C. L. 1988. Effects of tillage on the spatial pattern of microsclerotia of Macrophomina phaseolina. Phytopathology 78: 217–221. Papavizas, G. C. 1977. Some factors affecting survival of sclerotia of Macrophomina

44 Physiological, genetic, and pathogenetic variability

phaseolina in soil. Soil Biol. Biochem. 9: 337–341. Purkayastha, S., Kaur, B., Dilbaghi, N. and Chaudhury, A. 2004. Cultural and pathogenic variation in the charcoal rot pathogen from clusterbean. Ann. Agri. Biol. Res. 9: 217–221. Purkayastha, S., Kaur, B., Dilbaghi, N. and Chaudhury, A. 2006. Characterization of Macrophomina phaseolina, the charcoal rot pathogen of cluster bean, using conventional techniques and PCR-based molecular markers. Plant Pathol. 55: 106–116. Reyes-Franco, M. C., Hernandez-Delgado, S., Beas-Fernandez, R. Medina-Fernandez, M. Simpson, J. and Mayek-Pérez, N. 2006. Pathogenic and genetic variability within Macrophomina phaseolina from Mexico and other countries. J .Phytopathol. 154: 447–453. Short, G. E., Wyllie, T. D. and Bristow, P. R. 1980. Survival of Macrophomina phaseolina in soil and residue of soybean. Phytopathology 70: 13–17. Sinclair, J. B. and Backman, P. A., eds. 1989. Compendium of soybean diseases. 3rd ed. American Phytopathological Society, St. Paul, MN. 106 p. Su, G., Suh, S. O., Schneider, R. W. and Russin, J. S. 2001. Host specialization in the charcoal rot fungus, Macrophomina phaseolina. Phytopathology 91: 120–126. White, T. J., Bruns, T., Lee, S. and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a guide to methods and applications (eds. M.A. Innis, D.H. Gelfand, J.J. Sninisky and T.J. White). Academic Press, San Diego, USA: 315–322.

CHAPTER 3

Effect of rotation of cowpea (Vigna unguiculata) with Digitaria exilis and Pennisetum glaucum on Macrophomina phaseolina densities and cowpea yield

M. Ndiaye1, A. J. Termorshuizen2 and A. H. C. van Bruggen2

Macrophomina phaseolina, the causal agent of charcoal rot, causes great damage to cowpea in the Sahel. One of the few options to manage the disease is by cropping nonhosts that may reduce the soil inoculum below a damage threshold level. To test this, fonio (Digitaria exilis) and millet (Pennisetum glaucum) were cropped continuously for 3 years in plots with a natural infestation of 24–53 microsclerotia g-1 soil at the onset of the experiment. Next, a susceptible cowpea variety was grown to quantify disease incidence and severity on these soils. Fonio and millet both reduced microsclerotial densities in soils from the first year onwards. Reductions under fonio (81% after the 2nd year; 86% after the 3rd year) were significantly stronger than under millet (56% and 66% for the 2nd and 3rd year respectively). Fonio was not infected by M. phaseolina, while the root systems of millet had low densities of microsclerotia. Cowpea yielded significantly more hay and pods after 3 years of fonio than of millet. Cowpea yields and disease incidence (dead plants) could be explained well by pre- planting microsclerotial densities. We conclude that rotation of cowpea with a gramineous crop may lead to a relatively fast decline of inoculum density. In the case of a high inoculum density, fonio can be grown for three years to reduce M. phaseolina densities in soil.

Introduction Macrophomina phaseolina (Tassi) Goid. is a soilborne fungus causing charcoal rot and ashy stem blight on a wide range of plants in the world (Dhingra and Sinclair, 1978; Adam, 1986). In the Sahel, smallholder farmers grow cowpea and millet during the rainy season (June – September). Cowpea is host to M. phaseolina, but millet not (Ndiaye et al., in prep.). In the absence of hosts, the fungus survives in soil and host tissues in the form of microsclerotia, which are able to survive for 2–15 years in soil depending on environmental conditions, and whether or not the sclerotia are associated

1 AGRHYMET/DFR BP. 12625 Niamey, Niger; email: [email protected] 2 Biological Farming System Group, Wageningen University, Marijkeweg 22, 6709 PG Wageningen, The Netherlands; email: [email protected] and [email protected]

47 Chapter 3 with host residue (Cook et al., 1973; Short et al., 1980). Many factors have been reported to adversely affect the persistence of these propagules, for example soil moisture content (Dinghra and Sinclair, 1975), soil structure and depth (Short et al., 1980; Sharma et al., 1995; Young and Alcorn, 1984). Factors that adversely affect the survival of these propagules include also repeated freezing and thawing of soil, low carbon : nitrogen ratios in soil, high soil moisture content (Dhingra and Sinclair, 1975), high soil temperatures (Dhingra and Chagas, 1981; Lodha et al., 2002), and organic matter amendment (Israel et al., 2005). Several characteristics of the Sahelian zone particularly favour development of M. phaseolina, including low soil organic matter content, its associated limited soil microbial activity (Ouattara and Persaud, 1986), and low soil moisture content during the long off-season. In a preliminary study, we observed that under field conditions millet (Pennisetum glaucum) cv. HKP was not infected by M. phaseolina. However, the fungus was able to colonize millet roots saprophytically after physiological maturity. To avoid inoculum build-up, it may be better to lift millet plants at harvest time, a practice that is common in Sine Saloum (Senegal) (Diouf, 1990) and Maradi (Niger). Roots exposed to the sun dry quickly and limit colonization by the pathogen considerably. A significant reduction in nematode populations of Meloidogyne javanica and M. incognita race B was observed in pots cropped with fonio (Digitaria exilis) (Sarr and Prot, 1985). The authors suggested its use as rotation crop to manage nematode pests. The effects of fonio on other diseases are not well documented. In particular its effect on survival of inoculum of M. phaseolina is not known. Fonio is a popular crop in southern Senegal, Mali, Burkina Faso, and Niger. In these countries, it is cultivated in rotation with millet and cowpea. The crop is less important than millet in the Sahel with respect to cultivated areas and production. However, it has recently received more interest thanks to its flavour and nutritional qualities and to growing demands in developed countries and urban towns (Vodouhe et al., 2003). It may therefore be acceptable as rotation crop. This study aimed to investigate the susceptibility of millet and fonio to M. phaseolina and the effects of three years monocropping of millet (lifted from soil immediately after harvest) and of a local variety of fonio on soil propagules of M. phaseolina and disease incidence on cowpea in plots amended with compost.

Material and methods

Effect of M. phaseolina on millet and fonio (pot experiment) Three isolates of M. phaseolina (IS50, IS60 and IS100) isolated from soil (IS60 and IS100) and cowpea tissues (IS50) were multiplied on millet cv. HKP grains. Fifty g of

48 Effect of rotation grains were soaked during 24 h in demineralized water, the water excess was discarded, the soaked grains were placed in a 250 ml flask and sterilized at 121°C for 30 min. After cooling, the grains in the flask were inoculated by placing 6 5-mm diam discs cut out from a 3-d old fungal culture grown on PDA. The inoculated flasks were then incubated for 15 d at 30°C. After air-drying in a laminar air-flow cabinet, the colonized grain inoculum was used to infest sterile soil at a rate of 5% (dry weight) (Mayek-Pérez et al., 2001). Plastic pots of 500 ml, having 5 drainage holes and a cotton layer at the base were filled with the infested soil or with non/infested soil (control). Pots were thereafter sown with 10 seeds of fonio (local variety) or 9 seeds of millet cv. HKP and incubated in a screenhouse in a randomized complete block design with three replicates. Pots planted with three cowpea cv. Mouride seeds were used as susceptible check. Optimal soil moisture for fonio, millet and cowpea growth was afforded for 7 d after sowing. Then, the pots were irrigated after the seedlings underwent a moisture stress for one day. During the experimentation temperatures in the screenhouse were 30 ± 7°C. When plants were 45-days old, they were uprooted carefully; roots were washed with tap water and air-dried. Plant stand and d.w. biomass, and number of sclerotia g-1 root and lower stem tissues were recorded.

Experimental design and cultural practices in the field The study was conducted on an experimental field at the AGRHYMET centre (Niamey, Niger) that was naturally infested with M. phaseolina. The soil was a sandy soil (87% sand, 8.2% silt, 5.1% clay). The field was divided in 3 strata according to disease severity observed on cowpea at flowering in 1997 and soil inoculum density was recorded at planting in 1998: very high density (all plants dead and 53 microsclerotia g-1 soil), high (about 50% dead plants and 40 microsclerotia g-1 soil), and moderate (about 10% dead plants and 24 microsclerotia g-1 soil). In 1998, each stratum of about 12 m × 7 m was divided into four blocks, measuring 7 m × 3 m each; each of these blocks was divided into two plots of 10.5 m². Yearly before planting cereals, all blocks were amended with 6 tons of compost ha-1 and the top 15 cm was plowed with the aid of a bull. In 1998, 1999 and 2000, fonio and millet were planted randomly in 2 plots per block, resulting in 4 plots per crop per stratum. Millet was planted in 4 rows (1 m wide, with 10 hills per row and a within-row planting distance of 0.5 m) per plot. Fonio was broadcast in 3 × 3.5 m plots. In December 2001, the whole field was sown with cowpea cv. TVx 3236, known to be susceptible to M. phaseolina. Cowpea row-distance was 0.5 m and hill distance within rows was 0.25 m. Each hill was sown with 2 seeds. Weeding was done when needed and drip irrigation was performed weekly.

49 Chapter 3

Soil sampling and analyses In 1998 and 1999, soil samples were collected before planting to estimate the density of M. phaseolina. Four soil subsamples (0–30 cm) were taken with an auger (diam. 2.4 cm) from each stratum within a randomly chosen surface of 1 m2 according to a diamond pathway pattern (Mihail and Alcorn, 1987) and the subsamples were pooled. Three such composite samples were randomly taken in each stratum corresponding to a particular soil infestation level. In 2000 and 2001, one composite sample per plot was collected since plots were small. After thoroughly mixing the composite soil samples, a 5-g subsample per composite sample was analyzed by mixing 5 replicates of 1 g of soil and 100 ml of a Potato Dextrose Agar (PDA) medium amended with pentachloronitrobenzene (PCNB) (225 mg) and chloramphenicol (5 mg). One hundred ml of this medium was divided over 10 petri dishes and incubated for 8–10 days at 30°C after which the number of propagules of M. phaseolina was determined (Ndiaye et al., in prep.). Soil pH was measured yearly before planting using a pH-meter (Hi 8519N, Hanna Instrument, Inc., USA). Distilled water (25 ml) was added to 10 g soil of each subsample in a cup, stirred vigorously for 5 s and let stand for 10 min. The pH was read immediately (Soltner, 1990). The pHH20 was 6.8. Total N and total P contents of soil samples (0–30 cm) were determined using the methods described by Novozamsky et al. (1983; 1984). Three hundred milligrams air-dried, finely ground soil was digested using a mixture of H2SO4, Se and salicylic acid, and H2O2. After digestion, N and P were measured using segmented-flow analysis spectrometry. Bioavailable nutrients were determined using the methods described in Houba et al. (2000). Air-dried, ground soil samples were extracted for 2 h in 0.01 M CaCl2 using a 1:10 extraction ratio (w/v). In the extract NO3-N, NH4-N, total soluble N (Ntsoluble) and PO4-P were determined using segmented-flow analysis spectrometry; Na and K were determined using Flame Atomic Emission Spectrometry (Flame-AES). Total N and P content of the experimental field were 130 and 173 mg/kg soil respectively, which are relatively low concentrations for these elements. Plots were similarly low in concentrations of organic and inorganic elements (Table 1). The average organic matter content was 0.35% far less than the optimal content of 3–3.5 % for this type of soil. The C/N ratio was also low.

Estimation of millet and fonio colonization Colonization of millet and fonio tissue by M. phaseolina was assessed by destructively sampling 5 plants 30 and 60 d after planting (at the seedling stage and at physiological maturity, respectively). In addition, two months after harvesting, roots and stem residues

50 Effect of rotation

Table 1. Soil nutrient content of experimental plots.

Nutrient content (mg/kg soil)

Ntsoluble Norg P-PO4 Na K Nt Ptot OM (%) C/N 12.3 1.8 0.8 4.2 39.4 129.5 173.2 0.35 11.6

of fonio and roots of millet that had been lifted onto the soil surface were randomly sampled and assayed for the presence of microsclerotia. The sampled plant tissues were washed with tap water, cut into small pieces, surface-sterilized in 0.8% NaOCl for 1 min, blotted dry with paper towels, placed in a paper bag and dried in an oven at 37°C for 7 d. Dried tissues were ground in a mixer mill (Retsch, GmbH and Co. Type MM2) for 4 min at 600 rotations min-1 and sieved through 180 and 45 µm screens. Three subsamples of 5 g from each block were mixed with 100 ml of PDA amended with 5 mg chloramphenicol and 225 mg PCNB, and plated as described above. M. phaseolina colonies were counted after 5–7 days incubation at 33oC. Proportions of emerged and dead cowpea plants were recorded 10 d after planting and at harvest. Dry pod and hay yields were measured at harvest. Evaluation of emergence, disease incidence and yield were based on the two central rows of each plot.

Statistical analysis Data were analyzed by means of the computer program Genstat® for Windows 8th Edition (IACR-Rothamsted, Harpenden, Hertfordshire, UK). The data were subjected to analysis of variance. Treatment means were separated by the DMRT test. Numbers of microsclerotia in plant tissues were analyzed after log transformation if necessary.

Results

Pot experiment

Screening for resistance of fonio and millet There were no significant differences in total and root biomass (d.w.) between inoculated and non-inoculated plants for fonio and millet. The plant stand was also not affected by the pathogen (data not shown). For cowpea the weight of roots and stem of inoculated plants was significantly (P < 0.001) lower (0.11 and 0.095 g /d.w. plant, respectively) than roots and stem of non-inoculated plant (0.28 and 0.16 g /d.w. plant, respectively). Species significantly (P < 0.001) affected the number of microsclerotia

51 Chapter 3

Table 2. Microsclerotial density in roots and stems of 90 fonio and 81 millet plants growing in artificially inoculated soil in a screenhouse. Eight plants of the susceptible check (cowpea) were also analyzed for microsclerotial density.

Microsclerotia density (cfu g-1 tissue) Species Root Stem Fonio 3.0 ± 3.71c2 1.0 ± 2.3c Millet 42 ± 21b 15 ± 5.9b Cowpea 965 ± 280a 787 ± 149a 1 Standard deviation. 2 Means within a column followed by the same letter are not significantly different at α= 0.05.

in tissues (Table 2). However, mean microsclerotia population was low in both fonio and millet tissues.

Field experiments

1998 – 2000 crops. Millet and fonio emergence, plant stand, and growth were not affected by microsclerotial densities of M. phaseolina in the three strata, confirming that these crops are nonhosts. The density of microsclerotia in soil declined during the three years these nonhosts were grown. In all years, the reduction was significantly greater for fonio than for millet (P < 0.01). Millet did not significantly reduce the density of microsclerotia in soil for stratum 3 with a high initial inoculum density (Fig. 1). There were significant linear relationships between the density of microsclerotia in soil and the number of years cropped with fonio or millet (Table 3). However, at very high soil inoculum density, there was no linear relation between microsclerotial density and the number of years of millet cultivation.

Tissue and residue colonization. No propagules of M. phaseolina were found in fonio tissues and residues. Living tissues of millet were also not infected. However, two months after harvesting, millet roots showed a slight colonization that increased with soil inoculum density: 16 ± 13, 32 ± 12, and 40 ± 5 microsclerotia g-1 of dry root tissue in the moderately, highly and very highly infested soil, respectively.

52 Effect of rotation

Fig. 1. Evolution of microsclerotial soil inoculum density of Macrophomina phaseolina over 4 years in three plots with moderate (Stratum1), high (Stratum2), and very high (Stratum3) inoculum density after cropping cowpea in 1997 (before planting cereals in 1998), and after cropping the nonhosts fonio and millet in 1998, 1999, and 2000, and before cowpea in 2001. Vertical lines indicate the standard error.

Table 3. Regression equations for changes in soil microsclerotia over time (years) during cultivation of millet and fonio at 3 different levels of soil infestation. Soil inocula were recorded yearly before planting crops from 1998 to 2001.

Soil Crop Regression Constant Year Regression infestation species equations (P > t) 1 (P > t) (P > F) level Moderate Fonio 27.38 – 6.79x < 0.001 < 0.001 < 0.001 Millet 30.12 – 6.13x < 0.001 < 0.001 < 0.001 High Fonio 44.35 –11.29x < 0.001 < 0.001 < 0.001 Millet 52.00 –12.52x < 0.001 < 0.001 < 0.001 Very high Fonio 61.85 –13.56x < 0.001 < 0.001 < 0.001 Millet 52.10 – 3.27x < 0.001 0.28 0.28 1 Probability of a larger t or F value than expected under the null hypothesis.

53 Chapter 3

Cowpea crop in 2001. Analysis of variance for soil sclerotia of M. phaseolina at the beginning of the season, plant survival, and yield of cowpea indicated that effects of previous crops and the interaction between Crop and Stratum were significant (Table 4). The overall population density of M. phaseolina at planting in 2001 was significantly lower in moderately and highly infested soils (Strata I and II) (5 microsclerotia g-1 dry soil) than in very highly infested soil (26 microsclerotia g-1 dry soil). However, soil microsclerotia were three times more in plots cropped with millet than in plots with fonio at the stratum III (Table 5). There was a clear effect of initial inoculum density on disease incidence and yield of cowpea (Table 5). The effect of millet and fonio on soil inoculum density (Fig. 1) was clearly reflected in cowpea yields and pre-cowpea soil inoculum density. There was no effect of cropping history on emergence of cowpea. There was a highly significant negative correlation between microsclerotial density and the cowpea plant stand (r = –0.90; Fig. 2a) and a positive correlation between plant stand and grain yield (r = 0.68; Fig. 2b).

Table 4. Mean squares from the ANOVA table for soil density of Macrophomina phaseolina (number / g dry soil) at planting of cowpea, germination (%), dead plants (%), and pod and hay yield of cowpea (kg / ha), in 2001 after 3 years of non-host cropping with fonio or millet.

Mean squares

Source of DF Soil Germination Dead plants Grain yield Hay yield variation microsclerotia

Block 3 9.90 ns 13.72 ns 18.30 ns 22308 ns 187082

Crop 1 624.24*** 363.48*** 593.02*** 336382*** 3195346***

Stratum 2 1167.66*** 1324.10*** 267.04* 206834* 1933889**

Crop × 2 315.72** 137.71** 315.36*** 139160** 1102759* Stratum

Residual 15 30.68 18.71 23.02 20823 202236

Significant levels: ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns = not significant at α = 5 %.

54 Effect of rotation

Table 5. Viable sclerotia, plant emergence, percentage of dead plants at harvest, and dry pod and hay yield of cowpea in fields cropped three consecutive growing seasons with fonio or millet. The soil infestation levels in 1998 were 24, 40 and 53 microsclerotia per gram dry soil in moderately, highly and very highly infested soils respectively.

Sclerotia/g soil Emergence Dead plants Grain yield Hay yield before cowpea (%) (%) (kg/ha) (kg/ha) Crop Millet Fonio Millet Fonio Millet Fonio Millet Fonio Millet Fonio Soil infestation Moderate 7b1c 3c 76b 85a 18c 5d 455bc 855a 1797bc 2643a High 5bc 5bc 76b 79ab 19c 19c 357bc 527b 1473cd 2278ab Very high 39a 13b 64c 75b 45a 27b 270c 409bc 982c 1519cd 1 Means within two adjacent treatments (millet-fonio) followed by the same letter are not significantly different at α = 5%.

Fig. 2. Relationship between plant stand (%) and soil microsclerotia (cfu g-1 soil) (A) and cowpea grain yield (kg ha-1) and plant stand (%) (B). Actual data for the soil microsclerotia were transformed by log10 (x + 1) before plotting.

55 Chapter 3

Discussion The field used for the present study contained very low concentrations of mineral nutrients and organic matter (0.35%) with a C/N ratio equal to 12. It is well documented that environmental stresses including nutrition affect severity of M. phaseolina (Cloud and Rupe, 1994) by weakening the host plant or altering its natural resistance. Moreover, low microbial activity in soils low in organic matter may contribute to survival of microsclerotia in soil (Soltner, 1990). A C/N ratio of 30–50 is optimal for soil microorganism activities (Soltner, 1990). This suggests that there was low biological activity in the soils used in this study. Rotations are among the oldest methods to manage soilborne plant pathogens. M. phaseolina is in this respect difficult to manage given its wide host range (Adam, 1986) and the high microsclerotial densities in soil that are often encountered after growing hosts such as bean (17–47 microsclerotia g-1 soil), cowpea (15–45), sorghum (13–37), and maize (11–45) (Songa and Hillocks, 1996). In the semi-arid areas of the Sahel, commonly grown hosts include cowpea, sorghum, maize, peanut and other legume crops. Alternate cropping of cowpea and millet is common practice in the Sahel. This type of rotation is favorable to rapid build up of M. phaseolina, except when millet plants are lifted, including their root systems, which is common practice in the peanut basin of Senegal (Diouf, 1990). Although sorghum and maize were less susceptible than leguminous hosts, these gramineous crops still resulted in increase in density of M. phaseolina (Songa and Hillocks, 1996). A considerable reduction of 75–95% in the counts of M. phaseolina was achieved by biofumigation of irrigated soil at 46–53°C for 15 days after amending with cruciferous residues (Lodha et al., 1997). This rapid decline in soil inoculum was attributed to the combined effect of sub-lethal soil temperatures and toxic cruciferous residues. However, cruciferous crops are not suited to the Sahelian region. In the present study, cropping of fonio and millet both resulted in lower inoculum densities, decreased numbers of dead cowpea plants, and increased pod and hay yields. The effect of fonio was clearer than that of millet, which was likely due to the partial colonization of dead organic matter of millet. Higher colonization of millet root systems with microsclerotia of M. phaseolina has been observed (Ndiaye et al., in prep.) when the roots were left in soil. Here, root systems were uprooted and left on the soil surface exposed to the sun. In the present study we achieved 87–94% reduction in soil populations of M. phaseolina after three years monoculture of fonio. Fonio clearly is a nonhost to M. phaseolina under field conditions, so, it would be advisable to include it in the rotation with cowpea to prevent build up of populations of M. phaseolina. Survival of M. phaseolina in uncropped soil was investigated by Short et al. (1980) and Songa and Hillocks (1996). After burial of soybean residues with

56 Effect of rotation microsclerotia in a soil in Missouri, densities of M. phaseolina first increased for about half a year in fallow soil, and then decreased in the next half year (Short et al.,1980). In Eastern Kenya, densities of M. phaseolina also first increased and then decreased in fallow soil after addition of a mixture of infected residues of various crops to soil. The increase lasted only 3 months and the decrease 3–6 months (Songa and Hillocks, 1996). The persistence of the inoculum of M. phaseolina was attributed to the presence of weed hosts in the fallow plots. So, an additional aspect of cropping fonio is the suppression of weeds that can serve as host for M. phaseolina (Young and Alcorn, 1984; Songa and Hillocks, 1996). Cowpea grains and hay yields were significantly affected by preceding crop and soil infestation level of M. phaseolina. Yield advantages due to the treatments were, however, modest in comparison with the 1.2 t/ha potential grain yield of the variety used (INRAN, 1986), but acceptable as far as yield under farm conditions (200–400 kg/ha) is concerned. Heavy yield losses owing to plant death at flowering compelled farmers to abandon growing of cowpea in the Louga, Bambey regions of Senegal and Gabougoura in Niger. Monocropping of cowpea or biannual rotation of cowpea and millet, and intercropping of cowpea and millet contributed to the damage caused by M. phaseolina in these areas. Our data suggest that acceptable control of charcoal rot of cowpea at high inoculum densities in adverse Sahelian environments, can be achieved partly through use of nonhosts as fonio and (to a lesser degree) millet. Farmers could plant fonio or millet continuously for three years to reduce soil inoculum of M. phaseolina to a level safe for cowpea production under conditions of moderate and high soil infestation (Fig. 1). In case of very high soil infestation, millet rotation is not efficient for a rapid reduction of soil inoculum, but fonio can reduce inoculum to acceptable levels within four years of monocropping. However, monocropping with these grain crops may invoke other disease problems. Thus, more research is needed to find additional crops that are non hosts to M. phaseolina. In addition, alternative management strategies, such as increasing soil organic matter contents with compost, will need to be developed, as charcoal rot can only be controlled by an integrated management approach.

References Adam, T. 1986. Contribution à la connaissance des maladies du niébé (Vigna unguiculata (L.) Walp.) au Niger avec mention spéciale au Macrophomina phaseolina (Tassi) Goïd. Université de Renne I. Thèse de doctorat. 117 p. Cloud, G. L. and Rupe, J. C. 1994. Influence of nitrogen, plant growth stage, and environment on charcoal rot of grain sorghum caused by Macrophomina

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phaseolina (Tassi) Goid. Plant Soil 158: 203–210. Cook, G. E., Boosalis, M. G., Dunkle, L. D. and Odvody, G. N. 1973. Survival of Macrophomina phaseoli in corn and sorghum stalk residue. Plant Dis. Rep. 57: 873–875. Dhingra, O. D. and Chagas, D. 1981. Effect of soil temperature, moisture, and nitrogen on competitive saprophytic ability of Macrophomina phaseolina. Trans. Br. Mycol. Soc. 77: 15–20. Dhingra, O. D. and Sinclair, J. B. 1975. Survival of Macrophomina phaseolina sclerotia in soil: Effect of soil moisture, carbon: nitrogen ratio, carbon sources, and nitrogen concentrations. Phytopathology 65: 236–240. Dhingra, O. D. and Sinclair, J. B. 1978. Biology and pathology of Macrophomina phaseolina. Imprensa Universitaria, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil 166 p. Diouf, M. 1990. Analyse de l’élaboration du rendement du mil (Pennisetum typhoides Stapf. et Hubebb.). Mise au point d’une méthode de diagnostic en parcelles paysannes. Thèse de Doctorat de 3ème cycle, Institut National Agronomique Paris-Grinon. 227 p. Houba, V. J. G., Temminghoff, E. J. M., Gaikhorst, G. A. and van Vark, W. 2000. Soil analysis procedures using 0.01 M Calcium Chloride as extraction reagent. Commun. Soil Sci. Plant Anal. 31: 1299–1396. INRAN (Institut National de Recherches Agronomiques du Niger) 1986. Catalogue des variétés de mil, sorgho, niébé et arachide recommandé au Niger; INRAN ed.. Niamey, Niger. 86p. Israel, S., Mawar, R. and Lodha, S. 2005. Soil solarization, amendments and biocontrol agents for the control of Macrophomina phaseolina and Fusarium oxysporum f. sp. cumini in aridisols. An. Appl. Biol. 146: 481–489. Lodha, S., Sharma, S. K. and Aggarwal, R. K. 1997. Solarization and natural heating of irrigated soil amended with cruciferous residues for improved control of Macrophomina phaseolina. Plant Pathol. 46: 186–190. Lodha, S., Sharma, S. K. and Aggarwal, R. K. 2002. Inactivation of Macrophomina phaseolina during composting and effect of composts on dry root rot severity and on seed of clusterbean. Eur. J. Plant Pathol. 108: 253–261. Mayek-Pérez, N., López-Castañeda, C., Gonzales-Chavira, M. Garcia-Espenosa, R., Acosta-Gallegos, J., Martinez de Vega, O. and Simpson, J. 2001. Variability of Mexican isolates of Macrophomina phaseolina based on pathogenesis and AFLP genotype. Physiol. Molec. Plant Pathol. 59: 257–264. Mihail, J. D. and Alcorn, S. M. 1987. Macrophomina phaseolina: Spatial patterns in a cultivated soil and sampling strategies. Phytopathology 77: 1126–1131.

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Novozamsky, I., Houba, V. J. G., Temminghoff, E. and van der Lee, J. J. 1984. Determination of total N and total P in a single soil digest. Neth. J. Agric. Sci. 32: 322–324. Novozamsky, I., Houba, V. J. G., van Eck, R. and van Vark, W. 1983. A novel digestion technique for multi-element plant analysis. Commun. Soil Sci. Plant Anal. 14: 239–249. Ouattara, M. and Persaud, N. 1986. Soil and water constraints and adaptations to these constraints by farmers in rainfed cereal production. In Proceedings Niger Sorghum and Millet Workshop, 96–103 (Eds. J. D. Axtell and J. W. Clark). Purdue University, East Lafayette, Indiana 47907: INTSORMIL/IPIA. Sarr, E. and Prot, J. C. 1985. Pénétration et développement des juvéniles d’une souche de Meloidogyne javanica et d’une race B de M. incognita dans les racines de fonio (Digitaria exilis Stapf). Revue Nématol. 8: 59–65. Sharma, R. K., Aggarwal, R. K. and Lodha, S. 1995. Population changes of Macrophomina phaseolina and Fusarium oxysporum f. sp. cumini in oil-cake and crop residue-amended sandy soils. Appl. Soil Ecol. 2: 281–284. Short, G. E., Wyllie, T. D. and Bristow, P. R. 1980. Survival of Macrophomina phaseolina in soil and residue of soybean. Phytopathology 70: 13–17. Soltner, D. 1990. Les bases de la production végétale. Tom I. Le sol 18e édition. Collection Sciences et Techniques Agricoles, Le Clos Lorelle, Sainte-Gemmes- sur-Loire, 467p. Songa, W. and Hillocks, R. J. 1996. Legume hosts of Macrophomina phaseolina in Kenya and effect of crop species on soil inoculum level. J. Phythopathol. 144: 387–391. Vodouhe, S. R., Zannou, A. and Achigan, D. E. 2003. Actes du Premier Atelier sur la Diversité Génétique du Fonio (Digitaria exilis) en Afrique de l’Ouest. Conakry, Guinée, du 04 au 06 Août 1998. Institut International des Ressources Phytogénétiques (IPGRI), Rome, Italie. 81p. Young, D. J. and Alcorn, S. M. 1984. Latent infection of Euphorbia lathyris and weeds by Macrophomina phaseolina and propagule populations in Arizona field soil. Plant Dis. 68: 587–589.

CHAPTER 4

Effects of compost amendment on charcoal rot (Macrophomina phaseolina) development of cowpea

M. Ndiaye1, A. J. Termorshuizen2, and A. H. C. van Bruggen2

Macrophomina phaseolina is a destructive pathogen that causes charcoal rot of cowpea and other dicotyledonous crops in the semi-arid areas of the Sahel. Chemical management is not feasible in conditions of subsistence farming, and the plurivorous nature of the fungus limits the effectiveness of some cultural methods. This study was undertaken to determine the effects of composting on the survival of M. phaseolina and of soil application of compost without and with the biocontrol agent Clonostachys rosea on inoculum density of M. phaseolina and on cowpea production. Composting cowpea residues heavily infected with M. phaseolina raised the temperature in the heap to 52–60°C and completely destroyed the microsclerotia of M. phaseolina. Amendment of compost to planting holes significantly suppressed charcoal rot disease. Among the doses tested, 6 tons of compost gave the smallest Area-Under-the-Disease- Progress-Curve (AUDPC) and the highest cowpea production. Amendment with 50 kg NPK ha-1 reduced the disease and increased the yield even further. Among the biocontrol agents, two isolates of C. rosea completely overgrew the pathogen in dual culture. There was a sharp reduction in AUDPC and number of microsclerotia in cowpea tissues and a marked enhancement in grain yield when compost amended with C. rosea was added to the planting hole. The best treatment was obtained when both isolates of C. rosea were added together to the compost.

Introduction Cowpea (Vigna unguiculata (L.) Walp.), a valuable source of proteins for humans and animals, is the most important pulse crop in the Sahel, with nearly 12.5 Mha yr-1 (Singh et al., 1997). About 64% of the area cultivated to cowpea is located in West and Central Africa, the most important areas being in Nigeria (5.3 Mha and 2.3 Mtons), Niger (3.5 Mha, 0.5 Mtons) and Senegal (0.2 Mha, 0.09 Mtons) (FAOASTAT, 2005). In the Sahelian zone of West Africa, crop growth is most limited by water and nutrient stress (Penning de Vries and Djitèye, 1982), which are important predisposing

61 Chapter 4 factors to infection by the fungal pathogen Macrophomina phaseolina, the causal agent of charcoal rot (Cook et al., 1973; Songa and Hillocks, 1996; Rose and Barthès, 2001). M. phaseolina is a soilborne plant pathogen with a very wide host range and high ability to survive as microsclerotia that are formed in senescing shoot tissues (Short et al., 1978; Mayek-Pérez et al., 2002). Since 1969, there have been 29 years of virtually continuous drought in the Sahel, which contributed to decreasing organic carbon content of soils, due mainly to continuous cultivation, animal overgrazing, low biomass production and termite activities (Hulme, 1992; Cisse and Hall, 2002; Wezel and Haigis, 2002). Charcoal rot has become increasingly important in cowpea and other crops (Paré, 1990; Adam, 1986, Ndiaye, 1995 technical report ISRA/CNRA Bambey). Narrow rotations with susceptible crops, low organic matter content associated with small microbial populations and moisture stress are responsible for the increasing incidence of charcoal rot (Dhingra and Sinclair, 1978). The average yields of cowpea in farmers' fields are very low (0.2–0.5 ton ha-1) (Cisse et al., 1995; Cisse et Hall, 2002). Charcoal rot decreases these yields even further with an average yield loss of 10%, which is equivalent to an annual loss of 30.000 tons or an estimated $ 146 millions only for Niger and Senegal (Adam, pers. comm.). In Niger, Macrophomina disease has become important on a variety of hosts apart from cowpea, including sorghum (Sorghum vulgare), groundnuts (Arachis hypogea), okra (Hibiscus esculentus), sesame (Sesamum indicum), Dolichos lablab and sorrel (Hibiscus sabdariffa) (Adam, 1986). Also in Senegal the pathogen occurs widely (Bouhot 1967; 1968). Current agricultural practices in the Sahel do not include any methods to manage charcoal rot. Crop rotation may alleviate the disease problems a little, but there are too few nonhosts available and the pathogen is very persistent. Moreover, highly susceptible cowpea cultivars are widely grown. There are some reports on the efficacy of seed treatments with fungicides against soilborne and seedborne pathogens including M. phaseolina (Singh et al., 1990; Gamliel et al., 2000), but subsistence farmers of the Sahel can not afford these. An alternative approach to charcoal rot control is by augmenting the nutritional status of the soil (Osunlaja, 1990; Israel et al., 2005). This can be done using compost which at the same time increases the water holding capacity of the very poor sandy soil. Disease suppressive properties of compost have been reported regularly (e.g. Hoitink and Boehm, 1999; Noble and Coventry, 2005; Termorshuizen et al., 2006), although most of the reports deal with potting mixes and not with arable soils. Where significant effects were reported on disease suppression of compost amendments to arable soils, very high amounts of compost had been applied (e.g. Coventry et al., 2005). Own unpublished field experiments with broadcasting of 6 tons ha-1 compost did not result in any effect on charcoal rot. Since the application of greater amounts of

62 Effect of compost amendment compost is not feasible due to its limited availability to smallholder farmers in West Africa, we here report on the prospects of applying compost in the planting holes, using mixes of compost with soil free of M. phaseolina. By doing so, the young plants, which are relatively susceptible to M. phaseolina (Islam et al., 2003), are not predisposed to infections. Such a locally-applied amendment corresponds with the traditional ‘Zaï’ technique practiced in Burkina Faso, where manure is applied in the seeding holes, which reduces the amount of manure required considerably (Rose et al., 1993). The effect of compost on disease suppression is thought to be through a combination of microbial competition, referred to as general suppression, and effects of specific antagonists through e.g. amensalism, hyperparasitism or induced resistance, generally referred to as specific suppression (Hoitink and Boehm, 1999). The reliability of compost as a disease suppressive substrate can be increased by combining it with the application of a biocontrol agent (Hoitink and Boehm, 1999). In previous laboratory experiments, isolates of Clonostachys rosea (Link. Fr.) Schroers, Samuels, Serfert and Gams (anam. Gliocadium roseum Bainier) obtained from local arable soils appeared effective in reducing in vitro growth of M. phaseolina. C. rosea is a naturally occurring antagonist being common in arid areas (Sutton et al., 1997). This mycoparasite has a broad host range and has been used successfully to control pathogens such as Fusarium oxysporum f. sp. pisi, F. solani f. sp. pisi, Mycosphaerella pinodes, Rhizoctonia solani, Sclerotinia sclerotiorum and Pythium spp. (Xue, 2003; Møller et al., 2003). In a field naturally infested with a complex of soilborne pathogens, C. rosea increased pea emergence by 19% and yield by 15% yearly during three years (Xue, 2003). Ravnskov et al. (2006) indicated that the influence of C. rosea on various soil organisms depended on the presence of organic matter. Therefore, our goal was to test whether the joint application of C. rosea with compost in the planting hole would result in effective biocontrol of M. phaseolina. Given the ubiquity of M. phaseolina, the survival of inoculum during the preparation of compost was tested as well. M. phaseolina can tolerate temperatures up to 50–52°C for 120 h in steam-sterilized soils, but the fungus is killed in 8 h at 55°C and in 10 min. at 60°C (Paharia and Sahai, 1970). The objectives of this research were: 1. to determine the effect of composting on survival of M. phaseolina in cowpea residues; 2. to assess the effect of compost on charcoal rot development and cowpea production; 3. to determine the effect of joint amendment of compost and the biocontrol agent C. rosea on charcoal rot severity and yield of cowpea.

63 Chapter 4

Material and methods

Overview Four field experiments were carried out: in the dry (Exp. 1) and wet (Exp. 2) seasons of 2003, the wet season of 2004 (Exp. 3) and the wet season of 2005 (Exp. 4). The effect of compost amendment to planting holes was assessed on charcoal rot development and on yield of cowpea in a field naturally infested with M. phaseolina. The biocontrol potential of 6 local isolates of C. rosea against M. phaseolina was tested in a lab and pot experiment in 2005. The combined effect of compost and two local isolates of C. rosea was studied in a field naturally infested with M. phaseolina in the wet season of 2005 (Exp. 4).

Location and weather All experiments were conducted at the Regional Centre AGRHYMET, Niamey, Niger. The soil of the experimental field was a sandy soil (sand 87%, clay 5%, organic matter content 0.4%, pH 7.3). For the results of the chemical analysis of the soil see chapter 5. The mean daily temperature and relative humidity (RH) of the dry season (April – June) experiment in 2003 were 33°C and 50%, respectively. The water needed for growth was supplied by drip irrigation. In the wet season (July – September) of 2003, the mean daily temperature was 29°C, RH 70% and rainfall 335 mm. During the experimental period (May – August) in 2004, these figures were 29°C, 70%, and 344 mm, respectively, and in 2005 (May – August) 30°C, 83%, and 531 mm, respectively.

Microorganisms To study the survival of M. phaseolina during composting, naturally infected cowpea stems densely occupied with microsclerotia of M. phaseolina were collected in the field. For monitoring the viability of sclerotia in the composting pit and for evaluation of colonization of cowpea tissue with M. phaseolina, 150 mg (d.w.) of the milled tissues were mixed with 100 ml of Semi Selective for Macrophomina (SSM) medium and poured into 10 petri dishes. The SSM medium was prepared as follows: 1.5 ml of 0.525% NaOHCl, 1 ml of 0.5% chloramphenicol dissolved in 95% alcohol, and 10 ml of 2.25% quintozene (PCNB) were added to 100 ml of a PDA maintained at 55°C in a water bath. The medium was then poured in 10 petri dishes and incubated for 7–10 days at 33°C and the colonies of M. phaseolina were counted. For the dual culture tests with C. rosea, inoculum of M. phaseolina isolate GM3 (originally isolated from millet root and highly virulent on cowpea, and stored at 5°C on PDA) was used. For the pot experiment with C. rosea, inoculum of M. phaseolina was prepared as follows. Millet cv. HKP was soaked during 24 h in distilled water, the

64 Effect of compost amendment excess water was eliminated and 50 g of the soaked grains were autoclaved in a 250- ml flask (121°C for 30 min). After cooling, the grains were inoculated with M. phaseolina by placing 6 5-mm discs from a 3-d-old culture on Potato Dextrose Agar (PDA; Becton, Dickinson and Company Sparks, U.S.A.), and the flasks were incubated for 15 d at 30°C. After oven-drying at 37–40°C during 5 d, the colonized grains were milled, placed in plastic bags, sealed, and preserved at 4°C until use. Isolates of C. rosea (Table 1) were obtained from cowpea rhizosphere soil and roots or from rhizosphere soil of sorghum and stored at 4°C. Isolations were made from soil by serial dilution, and from roots by plating 150 mg milled dry roots on PDA as described above. Isolates were grown on PDA for two wk at 30°C, and spore suspensions were prepared by washing the culture with sterile water.

Table 1. Isolates of Clonostachys rosea used in this study.

Lab code1 Abbreviation Origin Year of isolation PiRNC1 Cr1 cowpea root 2004 PiRNC2 Cr2 cowpea root 2004 PiRNC3 Cr3 cowpea root 2004 PiRNC4 Cr4 cowpea root 2004 UARhS1 Cr5 rhizosphere soil of cowpea 2005 PiRhS2SO Cr6 rhizosphere soil of sorghum 2005 1 Indicates origin (location and crop or soil) and ID number of isolates.

Compost preparation A compost pit of 3 m long, 1.5 m wide and 0.20 m deep was dug under a mango tree. After having humidified the bottom, a 3-cm clay layer was laid out and then a 5-cm layer of cow manure and a 20 cm layer of 90% millet (Pennisetum glaucum) and 10% fonio (Digitaria exilis) straw (cut in about 10-cm-long pieces) were placed in the pit. The mixture was well moistened, compacted, flattened, and dusted with 500 g of ash. New layers of manure and straw were then added in the same way up to 0.80 m height. The heap thus obtained was covered with millet stalks. Every 15 d, the mixture was turned over, moistened and compacted. Temperature in the heap was recorded daily at 13 h GMT in the center and at three other locations of the heap 40–60 cm deep, using a thermometer (WWT LF91, Sartorius and Schott, the Netherlands). The temperature of the pit was obtained as the mean of the four records. The compost was considered to be mature when the temperature of the heap remained constant after turning over (Yacouba et al., 2001).

65 Chapter 4

Chemical analysis of compost Total N and total P content of compost samples was determined using the methods described by Novozamsky et al. (1983 and 1984). In short, 300 mg air dried, finely ground compost material was digested using a mixture of H2SO4, Se and salicylic acid, and H2O2. After digestion, N and P were measured using segmented-flow analysis spectrometry. Bioavailable nutrients were determined using the methods described in Houba et al. (2000). Air dried, ground compost samples were extracted for 2 h in 0.01

M CaCl2 using a 1:10 extraction ratio (w/v). In the extract NO3-N, NH4-N, total soluble N (Nt) and PO4-P were determined using segmented-flow analysis spectrometry, Na and K were determined using Flame Atomic Emission Spectrometry (Flame-AES). The concentration of various forms of nitrogen, phosphorus and potassium of the compost used in this study are shown in Table 2. Besides these nutrients, the sodium and organic matter contents are also listed.

Table 2. Nutrient content (mg/kg compost) and organic matter content (%) of the compost. Means of 2 determinations on 3 samples each.

Nt N Organic

N-NO3 N-NH4 soluble organic P-PO4 Na K Ntotal Ptotal matter 152.4 13.5 552.1 386.1 53.6 1100 4827 10419 2491 22.3

Effect of composting on survival of microsclerotia of M. phaseolina in cowpea Cowpea stems naturally infected with M. phaseolina were cut in pieces measuring 3–5 cm, and well mixed. On each of the 3 layers of the compost heap 15 nylon stocking bags containing 5 g of these small pieces of cowpea stem cuttings were placed. In addition, 45 bags with cowpea stem cuttings were exposed to ambient conditions next to the compost pit on top of the soil in the shadow. Each week, three bags were withdrawn from the compost pit, the cowpea stem cuttings dried at 40°C in an oven for 15 days, ground in a mill (Type MM2, Retsch, GmbH and Co KG, Haan, Germany) and assayed for viable sclerotia using the SSM medium for M. phaseolina described above. In the same way, samples of bags exposed to ambient air were analyzed weekly for viable sclerotia.

Biocontrol potential of C. rosea against M. phaseolina In vitro experiment. Six isolates of the antagonist were tested for their ability to inhibit

66 Effect of compost amendment the growth of M. phaseolina on PDA. Two 6-mm mycelial discs of actively growing cultures of M. phaseolina and C. rosea were placed side by side (1 cm apart) in the center of a three 9-cm diam petri dishes and incubated the plates at 30°C for 7 d (each treatment was repeated three times). The effect of the bioagents was determined by calculating the ratio of growth of the pathogen in the presence of C. rosea and that in the absence of C. rosea. Pot experiment. The two isolates of C. rosea that performed best in the in vitro assay were tested for their ability to reduce infection of cowpea by M. phaseolina in a pot experiment. The above-described millet inoculum of M. phaseolina and spore suspensions of isolates Cr1 and Cr2 of C. rosea were added to sterilized soil at a density of 5% (w/w; Mayek-Pèrez et al., 2001) and 108 cfu g-1 d.w. soil, respectively, and well mixed. Pots (1000 ml) were filled with the infested soil, watered, placed in the screenhouse over night and one surface-disinfested cowpea seed cv. Mouride was sown per pot. Treatments were M. phaseolina alone; M. phaseolina with C. rosea isolate Cr1; M. phaseolina with C. rosea isolate Cr2; M. phaseolina with C. rosea isolates Cr1 and Cr2 together (both at 108 cfu g-1 d.w. soil); and sterile millet grains with C. rosea isolates Cr1 and Cr2 (both at 108 cfu g-1 d.w. soil). Pots were incubated in a screenhouse in a randomized complete block design with three replicates. The mean temperature of the screenhouse was 30 ± 7°C. After 45 d, plants were uprooted carefully, the roots were washed with tap water, air-dried, and the number of sclerotia g-1 root and stem tissue were recorded.

Effect of compost amendments on soil inoculum of M. phaseolina and cowpea production Field experiments. The four field experiments were conducted in a naturally infested soil in a completely randomized block design with three replications. For the 2003 and 2004 trials the treatments consisted of four fertilization rates: 0 ton ha-1 (code c0), 3 -1 -1 -1 -1 tons ha (c3), 6 tons ha (c6) and 6 tons ha + 50 kg (N, P2O5, K2O) (15:15:15) ha (c6+), which corresponds to fertilizer units of 0, 52, 104, and 121 kg NPK ha-1, respectively. In 2005 the treatments consisted of 3 tons ha-1 of compost mixed with C. rosea isolates Cr1 or Cr2 (each at 108 cfu g-1 d.w. compost) or the combination of Cr1 and Cr2 (both at a density of 108 cfu g-1 d.w. compost). The control consisted of amendment with compost without inoculum of C. rosea. Treatments were applied in holes of 20 cm diam and 15 cm depth. Each plot consisted of 4 rows of 10 hills. Inter- row distance was 60 cm and between–hill distance 30 cm. The total plot size was 7.2 2 m . Compost (0, 54, 108, or 108 g + 1 g N, P2O5, K2O for treatments c0, c3, c6, and c6+, respectively) was mixed with 2.5 kg of soil (per hole), that had been steam sterilized for 8 h 3–4 d prior to planting. In Exp. 4 with C. rosea, the antagonist

67 Chapter 4 inoculum was mixed with the compost prior to mixing with sterilized sand and filling seeding holes. Sterilized sand was used to reduce infections of young plantlets. Each plot was irrigated with 83 liters water (corresponding to a soil moisture depth of 15 mm in the seeding line) once a day during 3 d by a drip irrigation system before cowpea cv. Mouride was planted (one seed per hole). When needed, plots were weeded with a hoe or watered by drip irrigation. In 2005 one insecticide application with 12 g ha-1 deltamethrin (Decis 12 EC, Aventis) was carried out against aphids (Aphis cracivora) at flowering stage of cowpea. Observations were made every 14 d from germination to harvest. Incidence, disease intensity, and time to death were recorded. Disease incidence was measured as the proportion of plants that were dead or severely wilted at each rating. The area under the disease progress curve (AUDPC) was used to measure disease progression. The standardized AUDPC was calculated as (Shaner and Finney, 1977):

n AUDPC += 2/ − ttxx , ∑i=1[]()ii −1 ()ii −1 in which n is the number of evaluation times, xi is the disease intensity at each evaluation time, and (ti–ti-1) is the time duration. Germination rate and plant survival, pod, grain and hay dry weight 75–90 days after planting were also recorded.

Statistics The computer program Genstat® for Windows, 8th Edition (IACR-Rothamsted, Harpenden, Hertfordshire, U. K.) was used for statistical analyses of studied parameters. All the data were subjected to analysis of variance (ANOVA) following a randomized complete block design. The treatment means were compared using Duncan’s Multiple Range Test at P = 0.05. Where needed data were transformed by log (x+1) before statistical analysis.

Results

Temperature evolution in the compost pit and viability of the sclerotia The highest temperature measured during composting was 64°C, which was recorded 8 d after preparation of the compost pit. The mean daily temperature in the compost pit exceeded 50°C for 22 d and 55°C for 13 d (data not shown). The mean weekly temperature in the compost pit was 46, 60, 56, and 52°C in the first, second, third, and fourth week respectively (Fig. 1)

68 Effect of compost amendment

70 Ambient 60 Compost 50 40 30 20

Temperature (°C) 10 A B 0 0 1 2 3 4 5 6 7 8 9 10 Week

Fig. 1. Weekly evolution of the temperature in the compost pit and ambient air (A) and the population changes of germinable sclerotia of M. phaseolina (log x+1) in composting and ambient conditions (B). Bars denote standard deviations.

The density of sclerotia in cowpea residues before composting was high (2691 sclerotia /g d.w.). During composting, the density of viable sclerotia decreased from week 3 onwards and the pathogen was undetectable from week 9 onwards. No significant changes were noticed in the number of sclerotia in crop residues exposed to ambient conditions (Fig. 1).

Disease development and yield of cowpea in 2003 and 2004 In the dry season of 2003, disease developed most prominently, as expressed by the relatively high AUDPC and low yields (Fig. 2). However, plants in the control plots were significantly (P < 0.021) more damaged than in the compost-amended plots except for AUDPC at 3 tons ha-1. Compost amendment consistently led to higher yields. The disease suppressive effect was significant (P = 0.003) at 3 tons ha-1 in 2004 and for the other two experiments at 6 tons ha-1 compost. At this last amendment level, AUDPC was 28, 56 and 45% lower as compared to the control for the 2003 dry, 2003 wet and 2004 experiment respectively. Yields were 66, 43 and 59% higher than the control, respectively. The effect of additional NPK was more evident for the yield than for the AUDPC over the three years of study (Fig. 2).

Effect of joint amendment of compost and Clonostachys rosea on Macrophomina disease development and cowpea production Among the tested isolates of C. rosea in dual culture, Cr1 and Cr2 exhibited the strongest antagonistic activity. In the dual culture test, these isolates completely overgrew the colonies of M. phaseolina within 7 days (Table 3). Green growth covered the pathogen colony. No inhibition zones between radial growths of the two fungi were observed.

69 Chapter 4

2003 dry 2003 wet 2004 wet

20 a ab 15 b a a b 10 ab b b

AUDPC b 5 b c

0 0366+ 0366+ 0366+ Compost dose (tons /ha)

2003 dry 2003 wet 2004 wet

1000 a 800 a

600 b b b c 400 a c a d Grain (kg/ha) b 200 c

0 0 3 6 6+ 0 3 6 6+ 0 3 6 6+ Compost dose (tons /ha)

Fig. 2. Effect of compost doses on area under disease progress curve (AUDPC) and cowpea grain yield per hectare in the 2003 dry and wet seasons and the 2004 wet season. 6+: 6 tons compost + 50 kg NPK ha-1.

In the pot experiment, the two isolates of C. rosea strongly and significantly suppressed M. phaseolina, resulting in healthy plants and low sclerotial densities (Table 4). Seedling appearance increased from 44% for the treatment with M. phaseolina alone to 89% for either Cr1 or Cr2 co-inoculated with M. phaseolina. Combining isolates Cr1 and Cr2 resulted in 98% emergence and the least infection (29 sclerotia per g tissue) (Table 4).

70 Effect of compost amendment

Table 3. Colony diameter of Clonostachys rosea and Macrophomina phaseolina plated side by side (1 cm apart) in the center of a 9-cm diam petri dish and extent of growth inhibition of M. phaseolina. Means of 3 replications.

Clonostachys rosea Macrophomina phaseolina Isolate Diameter (cm) of Diameter (cm) of Inhibition of growth (%) colony colony of Macrophomina colony Cr1 8.3a1 2.1b 98 Cr2 6.5b 3.3ab 80 Cr3 6.3b 3.3b 80 Cr4 4.4c 4.0b 69 Cr5 3.3c 5.5a 46 Cr6 5.9b 3.7b 74

1 Means in the column followed by the same letter are not significantly different at P < 0.05. In the control petri dishes the mean diameter of the colonies of M. phaseolina was 8.5 cm.

Table 4. Effect of Clonostachys rosea on the infection of cowpea seedlings by Macrophomina phaseolina. Means of 3 replications.

Treatment M. Seeding Seedling d.w. Root d.w. Sclerotia g–1 Sclerotia g-1 phaseolina C. rosea stand (%)1 (g plant-1) 2 (g/plant) 2 d. w. root2 d. w. stem tissues2 - - 100a3 0.84a 0.30a 0e 0a - Cr1Cr2 100a 0.86a 0.27a 0e 0a + - 44b 0.10c 0.00b 330a 176b + Cr1 89a 0.83a 0.20a 43c 0a + Cr2 89a 0.43b 0.20a 90b 0a + Cr1Cr2 98a 0.85a 0.22a 29d 0a 1 Observed 7 days after the onset of the experiment. 2 Observed 45 days after the onset of the experiment. 3 Means in the column followed by the same letter are not significantly different at P < 0.05.

In the field experiment, there was significantly more disease development in the plots amended with compost alone (control) than in the plots amended with both the

71 Chapter 4 compost and the Clonostachys isolates Cr1 and Cr1Cr2 (Fig. 3). Plant yield was significantly increased by the Clonostachys treatments but Cr1 performed better than Cr2 and joint inoculation of Cr1 and Cr2 resulted in the highest yields (Fig. 3).

1200 12 a a 1000 10

800 b b 8

600 c 6

400 c 4 AUDPC c Grain (kg/ha) Grain d 200 2 0 0 Control Cr1 Cr2 Cr1Cr2

Treatment

Fig. 3 Effect of soil amendment with compost combined with C. rosea on cowpea yield (grey bars) and charcoal rot development (white bars). Control: + compost, no antagonists; Cr1: + compost + Clonostachys isolate Cr1 (at 108 spores g-1 compost); Cr2: + compost + Clonostachys isolate Cr2 (at 108 spores g-1 compost); Cr1Cr2 = + compost + isolates Cr1 and Cr2 of Clonostachys (both at 108 spores g-1 compost). Letters above bars for the same variable indicate significant differences (P < 0.05) according to the Duncan‘s Multiple Range test.

Discussion In moist conditions both high (≥ 50°C) and low (-5 or 5°C) temperatures have been reported to adversely affect the survival and growth of M. phaseolina (Sheik and Ghaffar, 1987; Papavizas, 1977). The drastic reduction in sclerotial survival at low temperatures occurred when sclerotia were incubated at > 50% moisture holding capacity (MHC). In the present study, composting was effective at destroying microsclerotia in plant debris. Temperatures in the composting heap remained high (46–60°C) during the first 4 weeks of composting, which likely caused the sharp reduction in microsclerotia of M. phaseolina during composting. Indeed, Sheik and Ghaffar (1987) reported that in wet soil, viability of sclerotia of M. phaseolina was destroyed at constant temperatures > 55°C for 1 day or for 2 h per day for 14 days.

72 Effect of compost amendment

Turning the compost every 15 days allowed the exposure of all different parts of the compost heap to lethal temperatures. High moisture content (Dhingra and Sinclair, 1975; Sheik and Ghaffar, 1987; Sharma et al. 1995), release of volatile toxic compounds during composting (Lodha et al. 1997), and high levels of microbial populations are other factors that likely reduced the viability of the microsclerotia. The release of volatile toxic compounds such as ammonia has also been reported during the decomposition of pearl millet debris (Sharma et al., 1995), which was the main component of the compost heap. Charcoal rot development expressed by AUDPC was more severe during the dry than during the wet season, which likely was due to the high temperatures (33°C) and low relative humidity (50%), which both predispose hosts to infection by M. phaseolina (Mihail, 1989). Compost amendment significantly suppressed charcoal rot disease. Six tons of compost alone or amended with 50 kg ha-1 NPK improved cowpea production by 60% and suppressed charcoal rot significantly by 52% per season during 3 consecutive seasons. These doses probably reduced the number of root infections and subsequently led to reduction in wilt expression and plant mortality. Compost- amended sandy soils hold more water than non-amended treatments, which in turn can reduce the M. phaseolina population or its infection on the host plant due to enhanced antagonism or site competition. Husain and Ghaffar (1995) reported that, although root colonization of chickpea by M. phaseolina is greater at 40–50% MHC than at 10–20% MHC, wilting of plants occurred within 60 days at 10–20% MHC and 10–15 days later at 40–50% MHC. In our study, it was evident that application of compost increased the soil MHC and consequently reduced the drought stress of the cowpea plants. In the dry season, water uptake by roots of infected plants did not compensate water loss by plant transpiration. Consequently, plants faced moisture stress, which predisposed them to infection by M. phaseolina. In preliminary experiments it appeared that broadcasting 6 t ha-1 compost before sowing did not affect plant yield or disease severity at all. Generally, application of compost at higher rates leads to more disease suppression (Blok et al., 2000). To prevent early seedling infection, we used sterilized soil mixed with compost in the seeding hole. This is similar to the so-called ‘virgin soil technique’ introduced by Ko (1982). This method implies the use of pathogen-free soil around the plant. In the Sahelian environment this could be obtained by the use of steam-sterilized soil, of any other soil free of the pathogen, for example after solarization of a small soil surface (since smallholder farmers cannot afford plastic for treatment of large fields), or of non-cropped dune sand. Although steam-sterilized soil was used, high infection by M. phaseolina was observed in the non-amended controls, indicating the importance of the compost addition.

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Several mechanisms of antagonism of pathogens by C. rosea, including mycoparasitism, nutrient competition, and antibiosis have been suggested (Sutton et al., 1997). Characterization of the antagonistic action of local isolates of the biocontrol agent demonstrated that the isolates did not form an inhibition zone in dual culture with the pathogens. The antagonist acted predominantly by entwining (and parasitizing) the hyphae of M. phaseolina and probably also by space and competition for nutrients. These results are in accordance with the findings of Xue (2003) who demonstrated that the antagonistic capabilities of C. rosea isolate ACM941 were a result of mycoparasitism. In the field, joint amendment of 3 tons compost and C. rosea Cr1 or Cr1Cr2 was as effective as application of 6 tons compost + 50 kg NPK to increase yield and to reduce disease development of cowpea. According to Ravnskov et al. (2006), in addition to its antagonistic activity, C. rosea isolate Ik726 had a growth-promoting effect on tomato due mainly to increased P-solubilisation. Therefore the high yield of cowpea observed in the field experiment could be a result of a reduced cowpea infection by M. phaseolina and improved nutrient uptake by the plants. Our results indicate that in Sahelian sandy soil, good control and substantial increase of cowpea yield can be achieved by soil amendment with 6 tons compost ha-1. An even greater yield increase is achieved by soil amendment with 6 tons compost and 50 kg NPK ha-1 or 3 tons compost augmented by C. rosea. Two local isolates of C. rosea have good potential as biocontrol agents for M. phaseolina. The best control of the disease was obtained when the two isolates were combined. Further work is underway to test the effect of these isolates as seed treatment on the development of charcoal rot disease and production of cowpea.

References Adam, T. 1986. Contribution à la connaissance des maladies du niébé (Vigna unguiculata (L.) Walp.) au Niger avec mention spéciale au Macrophomina phaseolina (Tassi) Goïd. Université de Renne I. Thèse de doctorat. 117 p. Blok, W. J., Lamers, J. G., Termorshuizen, A. J. and Bollen, G. J. 2000. Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping. Phytopathology 90: 253–259. Bouhot, D. 1967. Etude du Macrophomina phaseoli sur arachide. Agr. Tropic. 22: 1165–1171. Bouhot, D. 1968. Le Macrophomina phaseoli sur les plantes cultivées au Sénégal. Agr. Tropic. 23: 1172–1181. Cisse, N. and Hall, A. E. 2002. Traditional Cowpea in Senegal, a Case Study. http://www.fao.org/AG/Agp/agpc/doc/publicat/cowpea_cisse/cowpea_cisse_e.htm.

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Cisse, N., Thiaw, S., Ndiaye, M. and Hall, A. E. 1995. Guide de production du Niébé. Fiches Techniques ISRA, vol 6 N° 2. 12 p. Cook, G. E., Boosalis, M. G., Dunkle, L. D. and Odvody, G. N. 1973. Survival of Macrophomina phaseoli in corn and sorghum stalk residue. Plant Dis. Rep. 57: 873–875. Coventry, E., Noble, R., Mead, A. and Whipps, J. M. 2005. Suppression of Allium white rot (Sclerotium cepivorum) in different soils using vegetable wastes. Eur. J. Plant Pathol. 111: 101–112. Dhingra, O. D. and Sinclair, I. D. 1978. Biology and pathology of Macrophomina phaseolina. Universidade Federal de Viçosa, Viçosa, Brazil. 166 p. Dhingra, O. D. and Sinclair, J. B. 1975. Survival of Macrophomina phaseolina sclerotia in soil: Effect of soil moisture, carbon : nitrogen ratio, carbon sources, and nitrogen concentrations. Phytopathology 65: 236–240. FAOSTAT, 2005. http://faostat.fao.org. Gamliel, A., Austerweil, M. and Kritzman, G. 2000. Non chemichal approach to soilborne pest management-organic amendments. Crop Protection 19: 843–847. Hoitink, H. A. J. and Boehm, M. J. 1999. Biocontrol within the context of soil microbial communities: a soil-dependent phenomenon. Annu. Rev. Phytopathol. 37: 427–446. Houba, V. J. G., Temminghoff, E. J. M., Gaikhorst, G. A. and van Vark, W. 2000. Soil analysis procedures using 0,01M calciumchloride as extraction reagent. Commun. Soil Sci. Plant Anal. 31: 1299–1396. Hulme, M. 1992. A 1951–80 global land precipitation climatology for the evaluation of general circulation models. Climate Dynamics 7: 57–72. Husain, T. and Ghaffar, A. 1995. Effect of soil moisture on the colonization of Macrophomina phaseolina on roots of chickpea. Pak. J. Bot. 27: 221–225. Islam, M. M., Sultana, K., Hussain, M. M., Mostafa, M. G. and Islam, M. R. 2003. Inoculation times with strains of Macrophomina phaseolina and Colletotrichum corchori on the seed yield contributing characters of late jute seeds. Pak. J. Plant Pathol. 2: 21–27. Israel, S., Mawar, R. and Lodha, S. 2005. Soil solarisaion, amendments and biocontrol agents for the control of Macrophomina phaseolina and Fusarium oxysporum f. sp. cumini in aridisols. Ann. Appl. Biol. 146: 481–491. Ko, W. H. 1982. Biological control of Phytophthora root rot of papaya with virgin soil. Plant Dis. 66: 446–448. Lodha, S., Sharma, S. K. and Aggarwal, R. K. 1997. Solarisation and natural heating of irrigated soil amended with cruciferous residues for improved control of Macrophomina phaseolina. Plant Pathol. 46: 186–190.

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Mayek-Pérez, N., Garcia-Espinosa, R., López-Castañeda, C., Acosta-Gallegos, J. A. and Simpson, J. 2002. Water relations, histopathology, and growth of common bean (Phaseolus vulgaris L.) during pathogenesis of Macrophomina phaseolina under drought stress. Physiol. Plant Pathol. 60: 185–195. Mihail, D. J. 1989. Macrophomina phaseolina: Spatio-temporal dynamics of inoculum and of disease in a high susceptible crop. Phytopathology 79: 848–855. Møller, K., Jensen, B., Andersen, H. P., Stryhn, H. and Hockenhull, J. 2003. Biocontrol of Pythium tracheiphilum in chinese cabbage by Clonostachys rosea under field conditions. Biocontrol Sci. Technol. 13: 171–182. Noble, R. and Coventry, E. 2005. Suppression of soil-borne plant diseases with composts: A review. Biocontrol Sci. Technol. 15: 3–20. Novozamsky, I., Houba, V. J. G., van Eck, R. and van Vark, W. 1983. A novel digestion technique for multi-element plant analysis. Comm. Soil Sci. Plant Anal.14: 239–249. Novozamsky, I., Houba, V.J.G., Temminghoff, E., and Van der Lee, J. J. 1984. Determination of total N and total P in a single soil digest. Neth. J. Agric. Sci. 32: 322–324. Osunlaja, S. O. 1990. Effect of organic soil amendments on the incidence of stalk rot of maize. Plant Soil 127: 237–241. Paharia, K. D. and Sahai, D. 1970. Effect of temperature on the viability of Macrophomina phaseoli from potato. Indian J. Microbiol. 10: 107–110 Papavizas, G. C. 1977. Some factors affecting survival of sclerotia of Macrophomina phaseolina in soil. Soil Biol. Biochem. 9: 337–341. Paré, D. 1990. Technique de quantification de l'inoculum et distribution géographique de Macrophomina phaseolina (Tassi) Goid. au Burkina Faso. Deuxième séminaire sur la lutte intégrée contre les ennemis des cultures vivrières dans le Sahel, 4 – 9 Janvier 1990. Bamako - Mali. CILSS, INSA, UCTR/PV. 12 P. Penning de Vries, F. W. T. and Djitèye, M. A. (Eds). 1982. La production des pâturages sahéliens. Une étude des sols, des végétations et de l'exploitation de cette ressource naturelle. Agric. Res. Rep. 918, PUDOC, Wageningen. 523 p. Ravnskov, S., Jensen, B., Knudsen, I. M. B., Bødker, L., Jensen, D. F., Karliński, L. and Larsen, J. 2006. Soil inoculation with the biocontrol agent Clonostachys rosea and the mycorrhizal fungus Glomus intraradices results in mutual inhibition, plant growth promotion and alteration of soil microbial communities. Soil Biol. Biochem. 38: 3453–3462. Rose, D. and Barthès, B. 2001. Organic matter management for soil conservation and productivity restoration in Africa: a contribution from Francophone research. Nutrient cycling. Agroecosystems 61: 159–170.

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Rose, E., Kabore, V. and Guenat, C. 1993. Le Zaï: fonctionnement, limites et amélioration d’une pratique traditionnelle africaine de réhabilitation de la végétation et de restauration de la productivité des terres dégradées de la région soudano-sahélienne. Cahiers ORSTOM série Pédologie 28: 159–174. Shaner, G. and Finney, R. E. 1977. The effect of nitrogen fertilization on the expression of slow-mildewing resistance in Knox wheat. Phytopathology 67:1051–1056. Sharma, S. K., Aggarwal, R. K. and Lodha, S. 1995. Population changes of Macrophomina phaseolina and Fusarium oxysporum f. sp. cumini in oil cake and crop residue – amended sandy soils. Appl. Soil Ecol. 2: 281–284. Sheik, A. H. and Ghaffar, A. 1987. Time-temperature relationships for the inactivation of sclerotia of Macrophomina phaseolina. Soil Biol. Biochem. 19: 313–315. Short, G. E., Wyllie, T. D. and Ammon, V. D. Quantitative enumeration of Macrophomina phaseolina in soybean tissues. Phytopathology 68: 736–741. Singh, B. B., Chamblis O. L. and Sharma, B. 1997. Recent advances in cowpea breeding. Pages 30–49 in Advances in cowpea research, edited by Singh, B. B., Mohan Raj, D. R., Dashiell, K. E., and Jakai, L. E. Copublication of International Institute of Tropical Agriculture (IITA) and Japan International Research Center for Agriculture Sciences (JIRCAS). IITA, Ibadan, Nigeria. Singh, S. K., Nene, Y. L. and Reddy, M. V. 1990. Influence of cropping systems on Macrophomina phaseolina populations in soil. Plant Dis. 74: 812–814. Songa, W. and Hillocks, R. J. 1996. The Charcoal rot in common bean with special reference to Kenya. Internat. J. Pest Manag. 42: 213–219. Sutton, J. C., Li, D. W., Peng, G., Yu, H., Zhang, P. G. and Valdebenito-Sanhueza, R. M. 1997. Gliocladium roseum: A versatile adversary of Botrytis cinerea in crops. Plant Dis. 81:316–328. Termorshuizen, A. J., Rijn, E. van, Gaag, D. J. van der, Alabouvette, C., Chen, Y., Lagerlöf, J., Malandrakis, A. A., Paplomatas, E. J., Rämert, B., Ryckeboer, J., Steinberg, C. and Zmora-Nahum, S. 2006. Suppressiveness of 18 composts against 7 pathosystems: Variability in pathogen response. Soil Biol. Biochem. 38: 2461–2477. Wezel, A. and Haigis, J. 2002. Fallow cultivation system and farmers‘ resource management in Niger, West Africa. Land Degrad. Dev. 13: 221–231. Xue, A. G. 2003. Biological control of pathogens causing root rot complex in field pea using Clonostachys rosea strain ACM941. Phytopathology 93:329–335. Yacouba, H., Morel, M. and Hounto, T. 2001. Valorisation par compostage de la biomasse de Pistia stratiotes issue de la station de lagunage de l’E. I. E. R. Sud Technol. 7: 40–47.

CHAPTER 5

Combined effects of solarization and organic amendment on charcoal rot caused by Macrophomina phaseolina in the Sahel

M. Ndiaye1, A. J. Termorshuizen2, A. H. C. van Bruggen2

The combined effects of soil solarization and amendment with millet residues or paunch contents on the survival of M. phaseolina and development of charcoal rot of cowpea were assessed in a naturally infested soil. In amended plots, solarization increased the soil temperature to 50°C for at least 4 h day-1 during June, leading to a significant reduction of soil inoculum of M. phaseolina by 44%. Paunch amendment (3 tons ha-1) caused 66% reduction of initial inoculum in the solar-heated plots while millet amendment did not enhance microsclerotia reduction in solarized plots. A slight reduction in microsclerotia of the pathogen was obtained in amended non-solar heated plots where the maximal temperatures were between 40–46°C. Significant reduction of charcoal rot disease and higher yield of cowpea were achieved in amended and/or solar-heated plots compared to the control. The lowest level of disease and the highest yield were observed in solarized plots that have been amended with paunch. Our results suggest that in the Sahelian zone the combination of solar heating and organic amendment can be a feasible strategy for managing charcoal rot disease and improving cowpea yield in fields with heavy infestations with M. phaseolina.

Introduction Cowpea is the main pulse crop of the semi-arid zones of the Sahel, but grain yields are usually as low as 200–300 kg ha-1 (Muleba and Ezumah, 1985). Many factors contribute to the low yield including shortage of nutrients, vulnerability to drought, and vulnerability to attack by pest and disease organisms (Mortimore et al., 1997). Potential solutions are available to partially solve the major constraints, except for the parasitic weed Striga and the fungal soilborne plant pathogen Macrophomina phaseolina, which causes ashy stem blight or charcoal rot of cowpea and many other crops (Frederiksen, 1986; Cisse et al., 1995). In Senegal and Niger, heavy soil infestations of M. phaseolina occur widely, usually resulting in total crop failure when cowpea is grown. The pathogen is able to persist in the absence of hosts for several

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years (Dhingra and Sinclair, 1977), and therefore additional measures are needed to manage this pathogen. Soil heating resulting in inactivation of multiple soilborne plant pathogens can be accomplished in warm climates by solarization. This involves covering the soil surface with a thin clear polyethylene tarp for several weeks to enhance accumulation of heat. It is used in regions with a warm climate to partially disinfest the soil. Soil solarization has been used to control Fusarium oxysporum f.sp. conglutinans (Ramirez-Villapudua and Munnecke, 1988), Verticillium dahliae (Melero-Vara et al., 1995; Pinkerton et al., 2000), Phytophthora cinnamomi (Pinkerton et al., 2000), Armillaria sp. (Otieno et al., 2003) and Macrophomina phaseolina (Chellemi et al., 1997; Lodha et al., 1997). Although soil temperatures attained by solarization may be sufficiently high to directly kill propagules of some of these pathogens situated in the upper soil layers, the efficacy declines with soil depth (Katan, 1981), and incorporation of additional suppressive factors is often necessary for improving the efficacy of soil solarization. In the Sahel during the dry season (October–June), the sun is an abundant source of energy, which can be used for heating soil-incorporated organic material in order to hasten material decomposition and to eliminate resting spores of fungal pathogens. Indeed, under suitable climatic conditions, solarization conducted for at least 4 weeks effectively controlled soilborne fungi and considerably reduced the population of weeds, nematodes and insects to a depth of 45–60 cm (Grünzweig et al., 1993; Gamliel et al., 2000). Combining cruciferous residues with solarization improved the control of Fusarium, M. phaseolina and Phytophthora spp. above that achieved by soil solarization alone (Ramirez-Villapudua and Munnecke, 1987, 1988; Lodha et al., 1997; Coelho et al., 1999). This was attributed to release of biotoxic volatiles, which are mainly responsible for the inhibition or reduction of soil-borne pathogens (Angus et al., 1994; Keinath, 1996; Mayton et al., 1996). The concentration of volatiles evolved was directly related to increase heating of soil (Gamliel and Stapleton, 1993b) and may also be related to partial anaerobiosis occurring under the plastic. Even in the absence of a strong temperature increase in the Netherlands, Blok et al. (2000) found strong and significant decline of Fusarium oxysporum f. sp. asparagi, Rhizoctonia solani, and Verticillium dahliae after incorporation into soil of 34–40 tons ha-1 f.w. grass followed with covering with an airthight plastic sheet. Soil inoculum reductions of F. oxysporum f. sp. cucumi and M. phaseolina by 70–80% have been reported after combining solarization with incorporation of 2.5 t ha-1 oil-cake or residues of mustard (Brassica juncea (Lodha et al., 1997; Lodha and Mawar, 2000). Amendments with nitrogen-enriched pearl millet residues significantly reduced the population of M. phaseolina within 45 days, but not that of Fusarium oxysporum (Sharma et al., 1995).

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In the Sahel, millet stalks are abundant and are nonhosts to M. phaseolina. The management of the paunch of slaughtered animals constitutes a problem in big towns. These materials (fresh or composted) could be used to increase the fertility of the poor sandy soils that characterizes this region and to contribute to the control of soil pathogens. The objective of this study was to investigate the individual and combined effects of soil incorporation of paunch of slaughtered animals, millet residues, and solar heating on survival of M. phaseolina and cowpea production.

Material and methods The study was conducted on an experimental field at the AGRHYMET Center (Niamey, Niger) containing a natural infestation level of M. phaseolina of 21 propagules g-1 soil. The soil was a sandy soil (87% sand, 8% silt, 5% clay) and was cultivated with groundnut (Arachis hypogea) cv. 55437 in 2002 and with a local, unnamed variety of sorrel (Hibiscus sabdariffa) in 2003 and 2004, both crops being susceptible to M. phaseolina. During the experimental period (May 2005) the weather conditions were: maximum air temperature 29.1–36.6°C, solar radiation 16.4–27.4 M J m-2 d-1, precipitation 550 mm.

Solarization and organic amendments The experimental field was subdivided in 3 blocks measuring 3.0×9.6 m each with 1.0 m margins in between. Each block was lengthwise divided in two plots and solarization was carried out to one randomly chosen plot per block. Each plot was divided in 4 subplots each measuring 2.4×3.0 m. On these subplots, the following treatments were carried out at random: (1) control (no organic amendment), (2) paunch content amendment, (3) millet residues amendment, (4) paunch and millet residues amendment. The experimental design was a split-plot with solarization and no solarization as main plots (repeated three times) and the four amendments as subplots. Millet residues, collected from farmer fields and chopped into 5-cm-long particles, and paunch content, obtained from the slaughterhouse of Niamey, were used as soil amendments (Table 1). The organic materials were incorporated at 3 tons dw ha-1 (= 1.08 kg subplot-1) to a depth of 10 cm. In the case of combined amendment of paunch content and millet residues, 3 tons ha-1 of both types of organic matter were added. Immediately after incorporation of the amendments, the soil was moistened to about 30 cm depth and plastic was applied on the three solarization plots on the same day. Plastic consisted of transparent, 50-µm-thick polyethylene (NETAFIMTM, Israel). The edges of the plastic were buried in order to reduce evaporation and to preserve the heat. The solarization treatments were maintained for 30 d. Soil thermometers were

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placed in the plots at 20 cm depth. Temperatures were recorded every 2 h from 7 to 24 h during 30 d.

Table 1. Nutrient contents (% of dry weight) of the two organic materials used as amendments.

Amendment total N (%) total P (%) K (%) Ca (%) Paunch content 1.08 0.37 0.60 0.63 Millet residues 0.57 0.15 0.50 0.48

Soil sampling and soil analysis Immediately before the experiment as well as after solarization, from each subplot 5 soil samples were withdrawn with an auger (diam. 1.5 cm) from the soil horizon 0–30 cm according to the diamond pathway pattern to estimate the density of M. phaseolina and the soil nutrient concentration. After thorough mixing of the soil samples, a 5-g subsample per composite sample was analysed by mixing 1 g of soil and 100 ml of a potato dextrose agar (PDA) medium amended with pentachloronitrobenzene (PCNB) (225 mg) and chloramphenicol (5 mg), divided over 10 petri dishes, incubated for 8–10 d at 30°C and the number of colonies of M. phaseolina was determined (Chapter 1).

Planting after solarization To determine the effect of the treatments on disease severity, 10 d after concluding the solarization treatments, plots were sown with cowpea cv. Mouride, which is highly susceptible to M. phaseolina. Before sowing, the seeds were soaked in 3% NaOCl for 5 min., washed with demineralised sterile water and dried on blotting paper. Seeds were sown in 50-cm-spaced rows, with a within-row space of 25 cm corresponding to six rows per plot. Weeding was done by hand as needed. After sowing, the trial was irrigated with drip irrigation. During the growth of cowpea, the rainfall ascertained the moisture need of the crop. Emergence was recorded 7 d after planting. The number of dead plants was recorded every 15 d and the Area-Under-the-Disease-Progress-Curve (AUDPC) calculated as follows according to Shaner and Finney, 1977:

n AUDPC += 2/ − ttxx , ∑i=1[( ii −1 ) ]( ii −1 )

in which n is the number of evaluation times, xi is the disease intensity at each evaluation times, and (ti – ti-1) is the time duration. Disease intensity is measured as the

82 Combined effects of solarization and organic amendment proportion of plants that are dead or severely wilted at each rating. Statistical Analyses. All the data were subjected to an analysis of variance four two criteria of classification using the computer program Genstat® for Windows 8th Edition Software developed by the Genstat Committee of the Statistics Department, IACR-Rothamsted, Harpenden, Hertfordshire AL52JQ). The treatment means were separated by the LSD (P = 0.05) test.

Results

Effect of solarization and organic amendment on soil temperature and fertility Maximum temperatures, recorded between 15 and 19 h, varied in the solarized treatments between of 48.3 and 50.1°C, which was about 10°C higher than in the non- solarized ones, where the range of maximum temperatures was 39.8–40.8°C. The effect of the organic amendments on soil temperature was negligible. Solarization did not affect soil pH, total N, total P and organic matter content, but available ammonium and nitrate increased significantly from 3.6 and 8.0 mg kg-1 to 9.8 and 13.6 mg kg-1 soil respectively (P = 0.034 and 0.024 respectively).

Effect of solarization and organic amendment on initial inoculum, charcoal rot development and yield of cowpea Solarization as well as amendment had a significant negative effect on soil inoculum of M. phaseolina and the AUDPC in cowpea plants grown on the treated soils and a significant positive effect on hay and grain yield of cowpea (Table 2). Solarization alone reduced the sclerotial soil inoculum density from 20.3 to 11.3 microsclerotia g-1 soil (44.3% reduction), while amendment of millet or paunch without solarization led to inoculum densities of 17.1 (15.8% reduction) or 13 (36.0% reduction) microsclerotia g-1 soil (Fig. 1). However, the effect on AUDPC was greater for the amendments alone than for the solarization alone: the millet and paunch amendments resulted in a reduction of AUDPC by 47.5 and 82.7% respectively, while solarization alone led to a reduction by 35.7% (Fig. 1). The best results were obtained for the combined treatments, which led to a reduction of soil inoculum by 46.3–65.8% and of AUDPC by 77.9–96.3% (Fig. 1). Cowpea yield was higher in solar-heated plots than in non-heated ones (Fig. 2). Solarization alone as well as millet and paunch amendment increased at least twice the hay and pod yields. Combination of solarization and organic matter increased yield more than did solarization or application of organic matter alone. The highest yield was achieved by combining paunch content with solarization or with a combination of paunch content, millet residue, and solarization.

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Table 2. Summary of the analysis of variance for soil population density, AUDPC, and pod and hay yield of cowpea at two heating levels and four amendment types.

Mean Square Source of variation df Soil inoculum AUDPC Hay yield Grain yield Main plot comparisons Solarization 1 296.807* 22.7647* 48053400* 1181757* Main plot error 2 5.982 1.4664 951905 14502 Subplot comparisons Amendment 3 35.904* 23.5867** 6733439** 638700** Solarization × Amendment 3 2.580 0.8674 1355204* 10469 Subplot error 12 2.291 0.6698 302873 17507

Significant levels: **: P < 0.01; *: P < 0.05.

Fig. 1. Effect of solarization for 30 d and/or organic amendment of millet or paunch residues (3 tons d.w. ha-1, added to 10 cm soil depth at day 0) on soil inoculum density of M. phaseolina and Area-Under-the-Disease-Progress-Curve (AUDPC) for cowpea plants grown after the solarization treatment. + = Solar heating.

84 Combined effects of solarization and organic amendment

Fig. 2. Effect of solarization for 30 d and/or organic amendment of millet or paunch residues (3 tons ha-1, added to 10 cm soil depth at day 0) on hay and grain yield of cowpea plants grown after the solarization treatment + = Solar heating.

Discussion The lethal temperature of M. phaseolina was investigated by Sheikh and Ghaffar (1987). In soils with high moisture content, microsclerotia were inactivated completely within 24 h at 50°C or higher. A strong decline occurred at 45°C, although a relatively small resistant fraction survived for at least 7 days. A strong decline also occurred at 40°C, but at 30 and 35°C an effect of temperature was not evident. In our study, solarization during 30 days reached on average 49°C as maximum temperature, about 10°C higher than the non-solarized treatments, and led to a significant reduction in inoculum of M. phaseolina. However, inactivation was lower than would be expected from the data provided by the field and laboratory experiments reported by Sheikh and Ghaffar (1984; 1987), who reported that solarization of naturally infested soil (5–7 sclerotia g-1) for one week increased soil temperatures at 20 cm depth from 30 to 41°C which led to a reduction of M. phaseolina-infected Vigna plants from 20 to 0%. Also in a loamy sand in India, where solarization led to a comparable increase of the soil temperature, inactivation of M. phaseolina was considerably higher: 100, 84 and 75% at soil depths of 5, 15 and 30 cm at an average soil moisture content after the solarization period of 2.7% (Israel et al., 2005). The relatively small effect of solarization attained by us may have been due to water leaching that likely has occurred in the coarse sandy soil, resulting in a dry top-layer. Dry soil conditions

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imply much more temperature resistance of microsclerotia of M. phaseolina. For example, at 50°C after 7 days of incubation reduction of inoculum of M. phaseolina was negligible in dry soils (moisture content 2–3%), and reduction was incomplete even at a temperature of 60°C (Sheikh and Ghaffar, 1987). In addition, dry soil conditions reduce the penetration of heat to deeper soil layers. Suboptimal effects of solarization alone have also been noted by other researchers (e.g. Gamliel et al., 2000). Notwithstanding the incomplete effects of solarization, addition of organic amendments at a rate of 3 tons dw ha-1 led to a strong increase in pathogen inactivation, and an even much stronger reduction in disease severity and increase in yield of cowpea planted after solarization. This combined effect has been noted before for various soilborne plant pathogens (Gamliel and Stapleton, 1993ab; Gamliel et al., 2000). It has also been noted for M. phaseolina, where solarization was combined with the amendment of 2.5 t ha-1 of Brassica juncea residues (Israel et al., 2005). In that study, however, the combined effect was less clear because of the high success rate of solarization alone on the soilborne inoculum of M. phaseolina (reduction >80%). Also in the absence of temperature increase, control effects have been described for cruciferous amendments on various soilborne pathogens due to the production of isothiocyanates during decomposition (Matthiessen and Kirkegaard, 2006) and for non-cruciferous amendments under nearly anaerobic conditions induced by plastic tarping (Blok et al., 2000). In this study, paunch amendment alone had an effect that was comparable to solarization alone with respect to inoculum reduction and yield increase. During decomposition of amendments high in nitrogen ammonia may be formed, which is highly toxic to multiple plant pathogens including M. phaseolina (Sharma et al., 1995; Lodha, 1995; Chun and Lockwood, 1985; Rodriguez-Kabana, 1986). In addition, there may have been an effect of the treatments on plant nutrition. + In the solarized and amended plots the concentration of ammonium (NH4 ) was higher than in non-solarized ones (9.75 vs 3.63 mg kg-1 soil). Although nitrogen fertilization has been reported to increase charcoal rot severity (Mote and Ramshe, 1980), the nitrogen concentrations in the untreated control may have been too low to allow for normal plant development, which may have predisposed the cowpea plants to infection by M. phaseolina. So, the fact that the effect was most evident for the organic amendment containing the highest nitrogen concentration, the paunch, could have been due to a combined effect of ammonia on the soil inoculum and an effect on plant nutrition. This is supported by the observation that the effect of solarization + paunch amendment on disease severity (expressed by AUDPC) was far greater (96.3% reduction in the paunch + solarization treatment) than that on soil inoculum level (65.8%). Finally, also the occurrence of sublethal temperatures in the solarization treatment likely have predisposed the pathogen to infection by resident antagonists

86 Combined effects of solarization and organic amendment next to the supposed action of ammonia (DeVay and Katan, 1991; Lodha et al., 2003). Our study demonstrated that under conditions where solarization alone does not provide sufficient control, the combination with organic amendments improves yields and reduces infection by M. phaseolina. High-N containing amendments may be most effective, such as the paunch used in this study. Solarization as well as application and incorporation of millet residues or paunch content in moistened soil can double cowpea production in poor naturally infested soil of the Sahelian zone and contribute to manage the paunch waste from slaughter houses in big cities.

References Angus, J. F., P. A., Gardner, Kirkegaard, J. A. and Desmarchelier, J. M. 1994. Biofumigation: Isothiocyanates released from Brassica roots inhibit growth of the take-all fungus. Plant Soil 162: 107–112. Blok, W. J., Lamers, J. G., Termorshuizen, A. J. and Bollen, G. J. 2000. Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping. Phytopathology 90: 253–259. Chellemi, D. O., Olson, S. M. Mitchell, D. J., Seeker, I. and McSorley, R. 1997. Adaptation of soil solarization to the integrated management of soilborne pests of tomato under humid conditions. Phytopathology 87: 250–258. Chun, D. and Lockwood, J. L. 1985. Reduction of Pythium ultimum, Thielaviopsis basicola, and Macrophomina phaseolina populations in soil associated with ammonia generated from urea. Plant Dis. 69: 154–158. Cisse, N., Thiaw, S., Ndiaye, M. and Hall, E. A. 1995. Guide de production de niébé. Fiches Techniques, Inst. Sénégalais de Recherches Agricoles: Vol. 6 n° 2, 22 p. Coelho, L., Chellemi, D. O. and Mitchell D. J. 1999. Efficacy of solarization and cabbage amendment for the control of Phytophthora spp. in north Florida. Plant Dis. 83: 293–299. DeVay, J. E. and Katan, J. 1991. Mechanisms of pathogen control in solarized soils. Pp 87–102 in Soil Solarization. Katan, J. and DeVay, J. E. eds. CRC Press Inc., Boca Raton, FL. Dhingra, O. D. and Sinclair, J. B. 1977. An annotated bibliography of Macrophomina phaseolina 1905–1975. Published cooperatively by Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil and the University of Illinois at Urbana– Champaign, IL. 244 p. Frederiksen, R. A. ed., 1986. Compendium of Sorghum Diseases. The American Phytopathological Society, St. Paul Minnesota, U.S.A. 82 p. Gamliel, A. and Stapleton, J. J. 1993a. Effect of chicken compost or ammonium phosphate and solarization on pathogen control, rhizosphere microorganisms, and lettuce growth. Plant Dis. 77: 886–891.

87 Chapter 5

Gamliel, A. and Stapleton, J. J. 1993b. Characterization of antifungal volatile compounds evolved from solarized soil amended with cabbage residues. Phytopathology 83: 899–905. Gamliel, A., Austerweil, M. and Kritzman, G. 2000. No-chemical approach to soilborne pest management - organic amendments. Crop Prot. 19: 847–853. Grünzweig, J. M., Rabinowitch, H.D. and Katan, J. 1993. Physiological and developmental aspects of increased plant growth in solarized soils. Ann. Appl. Biol. 122: 579–591. Israel, D., Mawar, R. and Lodha, S. 2005. Soil solarization, amendments and biocontrol agents for the control of Macrophomina phaseolina and Fusarium oxysporum f. sp. cumini in aridisols. Ann. Appl. Biol. 146: 481–489. Katan, J. 1981. Solar heating (solarization) of soil for control of soilborne pests. Annu. Rev. Phytopathol., 19: 211–36. Keinath, A. P. 1996. Soil amendment with cabbage residue and crop rotation to reduce Gummy Stem Blight and increase growth and yield of watermelon. Plant Dis. 80: 564–570. Lewis, J. A. and Papavizas, G. C. 1970. Evolution of volatile sulfur containing compounds from decomposition of crucifers in soil. Soil Biol. Biochem. 2: 239–246. Lodha, S. 1995. Soil solarization, summer irrigation and amendments for the control of Fusarium oxysporum f. sp. cummini and Macrophomina phaseolina in arid soils. Crop Prot. 14: 215–219. Lodha, S. and Mawar, R. 2000. Utilizing solar heat for enhancing efficiency of cruciferous residues for disinfesting soil borne pathogens from aridisols. Pp. 49–52 in Proc. Fifth International Symposium on chemical and non-chemical soil and substrate disinfestation. Gullino, M. L. et al. eds. Acta Horticulturae. Lodha, S. Sharma, S. K. and Aggarwal, K. K. 1997. Solarisation and natural heating of irrigated soil amended with cruciferous residues for improved control of Macrophomina phaseolina. Plant Pathol. 46: 186–190. Lodha, S. Sharma, S. K., Mathur, B. K. and Aggarwal, K. K. 2003. Integrating sublethal heating with Brassica amendments and summer irrigation for control of Macrophomina phaseolina. Plant Soil 256: 423–430. Matthiessen, J. and Kirkegaard, J. 2006. Biofumigation and enhanced biodegradation: Opportunity and challenge in soilborne pest and disease management. Crit. Rev. Plant Sci. 25: 235–265. Mayton, H. S., Olivier, C., Vaughn, S. F. and Loria, R. 1996. Correlation of fungicidal activity of Brassica species with allyl isothiocyanate production in macerated leaf tissue. Phytopathology 86: 267–271. Melero-Vara, J. M, Blanco-Lopez, M. A., Bejarano-Alcazar, J., and Jimenez-Diaz, R.

88 Combined effects of solarization and organic amendment

M. 1995. Control of Verticillium wilt of cotton by means of soil solarization and tolerant cultivars in Southern Spain. Plant Pathol. 44: 250–260. Mortimore, M. J., Sing B. B., Harris, F. and Blade, S. F. 1997. Cowpea in traditional cropping systems. Pp. 99–113 in Advances in Cowpea Research, Singh, B. B., Mohan Raj, D. R., Dashiell, K. E., and Jakai, L. E. eds. Copublication of International Institute of Tropical Agriculture (IITA) and Japan International Research Center for Agriculture Sciences (JIRCAS). IITA, Ibadan, Nigeria. Mote, U. N. and Ramshe, D. G., 1980. Nitrogen application increases the incidence of charcoal rot in rabi sorghum cultivars. Sorghum Newsletter 23: 129. Muleba, N. and Ezumah, H. C. 1985.Optimizing cultural practices for cowpea in Africa. pp. 291–298 in Cowpea Research, Production and Utilization, edited by Singh, S. R. and Rachie, K. O. John Willey and Sons, Chichester, U. K. Otieno, W., Termorshuizen, A., Jeger, M., Otieno, C. O. 2003. Efficacy of soil solarization, Trichoderma harzianum, and coffee pulp amendment against Armillaria sp. Crop Prot. 22: 325–331. Pinkerton, J. N., Ivors, K. L., Miller, M. L. and Moore, L. W. 2000. Effect of soil solarization and cover crops on populations of selected soilborne plant pathogens in Western Oregon. Plant Dis. 84: 952–960. Ramirez-Villapudua J. and Munnecke D. M. 1987. Control of cabbage yellows (Fusarium oxysporum f. sp. conglutinans) by solar heating of fields amended with dry cabbage residues. Plant Dis. 71: 217–21. Ramirez-Villapudua, J. and Munnecke, D. E. 1988. Effect of solar heating and soil amendments of cruciferous residues on Fusarium oxysporum f. sp. conglutinans and other organisms. Phytopathology 78: 289–295. Rodriguez-Kabana, R. 1986. Organic and inorganic nitrogen amendments to soil as nematode suppressants. J. Nematol. 18: 129–135. Shaner, G., and Finney, R. E. 1977. The effect of nitrogen fertilization on the expression of slow-mildewing resistance in Knox wheat. Phytopathology 67: 1051–1056. Sharma, S. K., Aggarwal, R. K. and Lodha, S. 1995. Population changes of Macrophomina phaseolina and Fusarium oxysporum f. sp. cumini in oil cake and crop residue-amended sandy soils. Appl. Soil Ecol. 2: 281–284. Sheikh, A. H. and Ghaffar, A. 1984. Reduction in viability of sclerotia of Macrophomina phaseolina with polyethylene mulching of soil. Soil Biol. Biochem. 16: 77–79. Sheikh, A. H. and Ghaffar, A. 1987. Time-temperature relationships for the inactivation of sclerotia of Macrophomina phaseolina. Soil Biol. Biochem. 19: 313–315.

CHAPTER 6

General discussion

The objective of this study was to identify different components for an integrated strategy for prevention and control of charcoal rot on cowpea caused by Macrophomina phaseolina. However, before being able to design control methods, more information was needed on the genetic variability among isolates of M. phaseolina, in particular with respect to virulence on different crop hosts and cultivars of cowpea. The present study demonstrated the variability in host susceptibility to M. phaseolina isolates from various Sahelian cropping systems, viz. continuous cowpea, cowpea-millet mixtures and cowpea-millet in rotation (Chapter 2). Although in the same cropping system isolates coexisted that were able to infect both cowpea and cereals, more isolates derived from continuous cowpea were highly pathogenic on cowpea, while the majority of isolates derived from cowpea in rotation with millet or cowpea intercropped with millet were more pathogenic on maize than isolates obtained from continuous cowpea. Four genotypes were distinguished in the cowpea/millet-based cropping systems by sequence analysis of the ITS region. Isolates of one of the genotypes (genotype 9) isolated from continuous cowpea cultivation systems were more virulent on cowpea than isolates from cowpea/millet intercropping or cowpea/millet rotation cropping systems. Changes in the population structure during continuous wheat cropping were also reported for the soilborne plant pathogenic fungus Gaeumanomyces graminis var. tritici. Genetically different strains of the fungus were present in the same field and a correlation between the severity of take-all disease and the frequency of some isolates was demonstrated (Lebreton et al., 2004; Lebreton et al., 2007). Therefore, strategies to manage charcoal rot should take into account not only the soil inoculum density but also the population structure of the pathogen as determined by cropping patterns and sequences. Due to persistent drought and limited input availability in the Sahel, millet and cowpea are almost the only food and cash crops grown as monoculture or mixed cropping systems. This results in severe reduction of cowpea yield due to heavy infestation of soils by M. phaseolina. In Chapter 3 of this thesis the suitability of selected cereal crops to act as rotation crops for soils infested with M. phaseolina was evaluated. Our results indicated that fonio, a scientifically neglected, but well adapted crop to Sahelian environment, is a non-host, millet a poor host and maize a susceptible host to M. phaseolina. Consequently, the build up of inoculum of M. phaseolina to damageable

91 Chapter 6 threshold levels can be delayed or prevented by including fonio in the crop rotation. Farmers would accept this recommendation, because fonio can grow successfully at 150–1000 mm annual rainfall isohyets and is still important to the food security of rural communities. Furthermore, the demand for fonio grains in developed countries and urban markets is increasing, the prices for fonio grain are higher than for other cereals and new equipment suitable for mechanical harvesting and threshing of fonio are being developed (Eyzaguirre and Thormann, 2003). Lack of an organized commercialization system and difficulties related to hand harvesting and threshing have been the main constraints of fonio production in West Africa until recently. Millet can be infected by the pathogen especially in heavily infested soils, but survival of soil inoculum can be lowered significantly by continuously cropping millet combined with lifting the millet plants with their roots at maturity. Lifting millet at maturity is a common practice in the Sine Saloum region of Senegal and is aimed at facilitating hand harvest operations and hastening stalk and root decomposition. This technique could be extended easily to farmers of other regions as a means of control of charcoal rot. Effects of crop rotation on soilborne pathogens, including those that form such persistent structures as sclerotia and chlamydospores are mostly based on their gradual dying-off in time if a suitable host is not present (Patil and Katan, 1997). Inoculum decline is often found in soils where nonhost crops preceded a susceptible host (Ruppel, 1985). Besides the simple exhaustion of nutrient reserves in the inoculum of soilborne pathogens in the absence of a suitable host, other mechanisms underlying rotation effects on plant diseases include root exudation of allelochemicals, toxic metabolites formed during the decomposition of organic residues, and production of compounds inhibitory for the pathogens or promotory for the competing microbiota (Khanh et al., 2005). Our results indicate that fonio not only prevents soil inoculum build up (because it is nonhost), but actively reduces soil microsclerotial densities of M. phaseolina after one year of cropping by 81% and after three consecutive crops by almost 100% in heavily infested soils. Cropping millet after cowpea reduces soil inoculum by only 56% the first year, due mainly to saprophytic colonization of millet roots at maturity. These results indicate that for selection of candidate crops in a rotation, attention should be paid to host susceptibility and persistence of the pathogen in crop residues. Besides reduction of the soil inoculum, fonio or millet in rotation with cowpea sharply reduced charcoal rot severity and increased cowpea yield even in heavily infested soils. Fonio, a grass-like crop, was cultivated continuously for three years on a field heavily infested by M. phaseolina, followed by a cowpea crop. This resulted in excellent control of charcoal rot on cowpea, expressed by a low AUDPC and a

92 General discussion significantly higher cowpea yield. Organic amendments are also known to affect survival of soil inoculum of M. phaseolina. The effect is more pronounced with organic materials with high-N content such as alfalfa or clover than organic matter with low C:N ratio such as cotton and wheat (Hakeem and Gaffar, 1977). Use of low C:N ratio organic materials stimulate the soil microflora, usually including biota that affect the viability of sclerotia. With compost, exogenous microorganisms are introduced in the soil, which may also contribute to disease suppression. Moreover, compost-amended sandy soils hold more water than non-amended treatments (Israel et al., 2005), which can lead to a reduction of the M. phaseolina population or to infection of the host plant due to enhanced antagonism or nutrient competition. However, very high amounts of compost are required to obtain significant disease suppression effects (Coventry et al., 2005). Such large quantities of compost are not available in Sahelian subsistence agriculture. In the present study, amendments of compost prepared from millet and fonio residues and manure reduced the severity of charcoal rot of cowpea, when it was applied in the planting hole (Chapter 4). A similar practice, but based on manure, is popular in Burkina Faso. The advantage of applying compost instead of manure is that composting of infected cowpea and millet residues during 9 weeks completely eliminated M. phaseolina propagules. Another way to effectively (66%) reduce soil inoculum of M. phaseolina and to improve soil fertility in the Sahel is by combining solarization and application of organic matters (Chapter 5). The organic materials tested were millet residues and paunch contents of slaughtered animals, materials that were relatively readily available to farmers. Numerous studies have been done to demonstrate the antagonistic potential of soil bacteria and fungi against M. phaseolina both in vitro and in vivo (Jana et al., 2000; Siddiqui and Shaukat, 2003; Srivastava et al., 1996). Chapter 4 of this study dealt with the potential of controlling charcoal rot of cowpea by biological means. The results indicate that in Sahelian sandy soil, good control and substantial increase of cowpea yield can be achieved by application of 3 tons ha-1 of compost augmented by C. rosea. Two local isolates of C. rosea had good potential as biocontrol agents for M. phaseolina in the Sahelian conditions. The best control of the disease was obtained when the two isolates were combined. Therefore this study points out a new perspective for the biological control of M. phaseolina in Sahelian environment using local isolates of C. rosea applied together with a small amount of compost. Use of cultural practices or resistant cultivars to reduce outbreaks caused by soilborne pathogens is the only feasible option for Sahelian smallholder farmers. Our results indicate that good control of charcoal rot of cowpea can be achieved by including fonio in the cowpea/millet rotation scheme, paunch amendment in

93 Chapter 6 combination with solar heating and amendment with compost augmented by C. rosea isolates. Solarization as well as application and incorporation of millet residues or paunch contents in moistened soil can double cowpea production in poor, naturally infested soil of the Sahelian zone and contribute to the management of paunch waste from slaughter houses in big cities. Apart from the increased yield and reduced disease severity, organic matter amendment can also improve soil fertility.

Recommendations for future research We developed four appropriate technologies very promising for the control of charcoal rot of cowpea in the Sahel, namely a change in crop rotation and application of low cost materials (plastic mulch for solarization, composted millet residues together with manure or paunch contents, and the biocontrol agent C. rosea applied in compost). We conclude that the disease pressure can only be reduced if various control measures are combined in an integrated management strategy. Therefore, future research should focus on developing an integrated strategy based on different combinations of control measures adapted to local conditions. It is necessary to calculate the costs and benefits for all feasible combinations.

References Coventry, E., Noble, R., Mead, A. and Whipps, J. M. 2005. Suppression of Allium white rot (Sclerotium cepivorum) in different soils using vegetable wastes. Eur. J. Plant Path. 111: 101–112. Eyzaguirre, P. and Thormann, I. 2003. Strategies for the conservation and use of fonio, an important but neglected crop of West Africa. Pp. 45–52; in: Actes du Premier Atelier sur la Diversité Génétique du Fonio (Digitaria exilis) en Afrique de l’Ouest, Vodouhe, S. R., A. Zannou et E. Achigan Dako eds. Conakry, Guinée, du 04 au 06 Août 1998. Institut International des Ressources Phytogénétiques (IPGRI), Rome, Italie. Hakeem, A. and Ghaffar, A. 1977. Reduction of the number of sclerotia of M. phaseolina in soil by organic amendments. Phytopathol. Z. 88: 272–275. Israel, D., Mawar, R. and Lodha, S. 2005. Soil solarization, amendments and biocontrol agents for the control of Macrophomina phaseolina and Fusarium oxysporum f. sp. cumini in aridisols. An. Appl. Biol. 146: 481–489. Jana, T. K., Srivastava, A. K., Csery, K. and Arora, D. K. 2000. Agglutination potential of Pseudomonas fluorescens in relation to energy stress and colonization of Macrophomina phaseolina. Soil Biol. Biochem. 32: 511–519. Khanh, T. D., Chung, M. I., Xuan, T. D. and Tawata, S. 2005. The exploitation of crop allelopathy in sustainable agricultural production. J. Agr. Crop Sci. 191:

94 General discussion

172–184. Lebreton, L., Gosme, M., Lucas, P., Guillerm-Erckelboudt, A. Y., Schoeny, A. and Sarniguet, A. 2007. Linear relationship between Gaeumanomyces graminis var. tritici (Ggt) genotypic frequencies and disease severity on wheat roots in the field. Env. Microbiol. 9: 492–499. Lebreton, L., Lucas, P., Dugas, F., Guillerm, A. Y., Schoeny, A. and Sarniguet, A. 2004. Changes in population structure of the soilborne fungus Gaeumannomyces graminis var. tritici during continuous wheat cropping. Env. Microbiol. 6: 1174–1185. Patil, J. and Katan, J. 1997. Effect of cultivation practices and cropping sytems on soilborne diseases. Pp. 377–396; in: Soilborne diseases of tropical crops; R. J. Hillocks and J. M. Waller eds. CAB INTERNATONAL, University Press Cambridge. Ruppel, E. G. 1985. Susceptibility of rotation to a root rot isolate of Rhizoctonia solani from sugar beet and survival of the pathogen in residues. Plant Dis. 69: 871– 873. Siddiqui, A. and Shaukat, S. S. 2003. Combination of Pseudomonas aeroginosa and Pochonia chlamydosporia for control of root-infecting fungi in tomato. J. Phytopathol. 151: 215–222. Srivastava, A. K. Arora, D. K., Gupta, S., Pandey, R. R. and Lee, M. 1996. Diversity of potential microbial parasites colonizing sclerotia of Macrophomina phaseolina in soil. Biol. Fertil. Soils 22: 136–140.

SUMMARY

Cowpea (Vigna unguiculata Walp.) is the most important pulse crop in the Sahel with nearly 12.5 million hectares per year and is a valuable source of protein for human and animal nutrition. About 64% of the area under cowpea is located in West and Central Africa. The most important cowpea growing areas are in Nigeria (about 4 million ha and 1.7 million tons), Niger (3 million ha, 0.3 million tons) and Senegal (0.12 million ha and 0.08 million tons). The most limiting factors for crop growth in the Sahelian zone of West Africa are water and nutrient stress. In Niger and Senegal, cowpea is grown mainly in the semiarid and arid zones, where the average annual rainfall is 250–500 mm. For small-scale farmers, the parasitic plant Striga, the insect pests cowpea aphid, cowpea weevil, cowpea flower thrips, Maruca pod borer, and hairy caterpillar and the soilborne plant pathogenic fungus Macrophomina phaseolina, causing charcoal rot bring about the most significant problems for the cultivation of cowpea, charcoal rot being one of the most destructive. The conditions of low levels of soil moisture leading to drought stress result in crops becoming more susceptible to infection by the pathogen. Charcoal rot is becoming increasingly important in several crops. Since 1981, charcoal rot has been spreading yearly in Niger in sorghum (Sorghum vulgare), groundnuts (Arachis hypogea), okra (Hibiscus esculentus), sesame (Sesamum indicum), lablab (Dolichos lablab) and sorrel (Hibiscus sabdariffa). Surveys in the main cowpea production areas in Niger, during four consecutive years (1982–1985) revealed that damage caused by M. phaseolina occurs throughout the country except in the district of Dosso in the Sudano-sahelian zone. The presence of M. phaseolina was already reported in the 1960’s in the majority of soils of Senegal. However, the fungus did not cause important damage on crops until 1987 when heavy yield loss due to charcoal rot damage was regularly observed in cowpea, but also sometimes on groundnut in the Louga zone of Senegal. The average cowpea yields in farmers' fields are low (0.2–0.5 ton/ha) and charcoal rot causes on average a yield loss of 10%, which is equivalent to 30.000 tons cowpea - an estimated value of $146 million for Niger and Senegal alone. M. phaseolina is sensitive to fungicides, and the application of fungicide to seed and soil can reduce fungal germination and infection. However, chemical control of this fungus is difficult and neither profitable nor advisable because the pathogen is seedborne and soilborne. Moreover, fungicides are too costly for subsistence farmers in the Sahel. Disease resistant or tolerant cultivars of cowpea against M. phaseolina are the most efficient control measures but tolerant /resistant cultivars are not yet

97 Summary available. Solarization, addition of organic matter, maintenance of high soil moisture, fumigation and use of biocontrol agents have shown to be potential methods for control of soilborne pathogens. However, there are no efficient control methods which can stand alone against charcoal rot. The disease pressure can only be reduced if different preventive control measures are combined in an integrated management strategy. In Chapter 2, the physiological and genetic variability and pathogenicity of isolates of M. phaseolina responsible for charcoal rot of cowpea are described. Isolates were collected from soil and infected tissues of different crop systems in Senegal and Niger. Isolates were characterized by their growth potential at different temperatures, their morphology on PDA medium, their virulence on three cowpea cultivars and sequence analysis of the ITS (internal transcribed spacer) region of the nuclear ribosomal DNA gene. A subset of 20 isolates was selected based on growth characteristics on PDA to study their pathogenicity on other crops besides cowpea. The cowpea cultivar Mouride with delayed leaf senescence was evaluated in naturally infested soil for resistance to M. phaseolina. Isolates of M. phaseolina differed to a limited extent in temperature optimum, genetic make-up, and pathogenicity to cereal crops. The grouping according to physiological and genetic traits did not coincide with that based on pathogenicity. However, for the first time, we showed some specialization in pathogenicity to cereal crops (except fonio (Digitaria exilis)) for isolates obtained from fields grown to millet next to cowpea. Field observations and in vitro studies indicated that fonio is a non-host to M. phaseolina, and millet a poor host, respectively. The influence of continuous cropping of these crops on soil inoculum density of M. phaseolina was studied under field conditions. The experiment was conducted in naturally infested fields in a randomized complete block design. The effect of three-year monocropping of fonio and millet on survival of microsclerotia of M. phaseolina was assessed in three plots with different soil inoculum levels. In the fourth year the effect of these practices on cowpea yield was also assessed (Chapter 3). Fonio was not infected by M. phaseolina, while the root systems of millet had low densities of microsclerotia. Cowpea yielded significantly more hay and pods after 3 years of fonio than after 3 years of millet. We conclude that rotation of cowpea with a gramineous crop may lead to a relatively fast decline of inoculum density. In the case of a high inoculum density, fonio can be grown for three years to reduce M. phaseolina densities in soil. A field experiment was conducted on the effect of compost on soil inoculum and symptom severity of charcoal rot (Chapter 4). The experiment was carried out in naturally infested farmers’ fields. The effect of 3 or 6 tons of compost and of 6 tons of compost supplemented with 50 kg NPK fertilizer ha-1, applied in planting holes, on

98 Summary charcoal rot was assessed for three consecutive years. Besides, the combined effects of 3 tons of compost ha-1 and the biocontrol agent Clonostachys rosea (108 CFU’s per g compost) on charcoal rot development and cowpea production were investigated. Our results indicated that in Sahelian sandy soil good control and substantial increase of cowpea yield can be achieved by soil amendment with 6 tons of compost ha-1. An even greater yield increase is achieved by soil amendment with 6 tons of compost and 50 kg NPK ha-1 or 3 tons ha-1 of compost augmented by C. rosea. In Chapter 5, the combined effects of soil solarization and amendment with millet residues and paunch contents on the survival of M. phaseolina and development of charcoal rot of cowpea were assessed in a naturally infested soil. The experimental design was a split plot with solarization as main plot and amendment as subplot. The organic materials were tested at 3 tons per hectare. In amended plots, solarization increased the soil temperature to 50°C for at least 4 h day-1 during June, leading to a significant reduction of soil inoculum of M. phaseolina by 44%. Paunch amendment (3 tons ha-1) caused 66% reduction of initial inoculum in the solar-heated plots while millet amendment did not enhance microsclerotia reduction in solarized plots. These observations demonstrated that under conditions where solarization alone does not provide sufficient control, the combination with organic amendments improves yields and reduces infection by M. phaseolina. High-N containing amendments may be most effective, such as the paunch used in this study. Solarization as well as application and incorporation of millet residues or paunch content in moistened soil can double cowpea production in poor, naturally infested soil of the Sahelian zone and contribute to the management of paunch waste from slaughter houses in big cities. This study indicated that M. phaseolina isolates from different cropping systems in the Sahel vary with respect to virulence and ability to cause infection on crop species and cultivars that are not highly susceptible to this pathogen. Among isolates of M. phaseolina, one genotype that was highly virulent on cowpea belonged to continuous cowpea cropping systems from Senegal. For the first time we found isolates of C. rosea in the Sahel that are efficient at controlling M. phaseolina. In addition we showed that including fonio in the rotation scheme of cowpea and millet, adding millet residues or paunch contents with or without solar heating and application of 3 tons ha-1 of compost augmented with C. rosea reduced charcoal rot disease and increased cowpea yield. These methods can be integrated into different combinations according to local conditions for managing charcoal rot. This integrated strategy based on feasible and low cost treatments could be easily adopted by farmers.

RÉSUMÉ

Le niébé (Vigna unguiculata (L.) Walp.) est la principale légumineuse cultivée au Sahel avec presque une superficie de 12,5 millions d’hectares par an. Il constitue une source de protéines appréciable pour l’alimentation humaine et animale. Environ 64% des superficies emblavées en niébé se trouvent en Afrique occidentale et centrale. Les principaux pays producteurs de niébé sont le Nigeria (environ 4 millions d’ha et 1,7 millions de tonnes), le Niger (3 millions d’ha, 0,3 millions de tonnes) et le Sénégal (0,12 millions d’ha et 0,08 millions de tonnes) en 2005. Les principaux facteurs limitant la croissance de la production dans la zone sahélienne de l’Afrique de l’Ouest sont la faible et la mauvaise répartition de la pluviométrie, la faible teneur en éléments nutritifs des sols et les nuisibles. Au Niger et au Sénégal, le niébé est cultivé principalement dans les zones semi-arides et arides où la moyenne pluviométrique annuelle est comprise entre 250–500 millimètres. Le striga, les pucerons, les bruches, les thrips des fleurs, le foreur de gousses (Maruca testularis), la chenille poilue (Amsacta monoleyi) et la pourriture charbonneuse (Macrophomina phaseolina) constituent les principales contraintes parasitaires à la production du niébé. Parmi ces contraintes, la pourriture charbonneuse constitue la maladie la plus destructive. Les conditions de faible humidité du sol qui provoquent des stress hydriques chez le niébé en développement augmentent sa sensibilité à l’infection de M. phaseolina. Les pertes de récoltes imputées à la pourriture charbonneuse devient de plus en plus importante dans plusieurs cultures. Depuis 1981, la pourriture charbonneuse est observée annuellement au Niger sur le sorgho (Sorghum vulgare), l’arachide (Arachis hypogea), le gombo (Hibiscus esculentus), le sésame (Sesamum indicum), la dolique (Dolichos lablab) et l’oseille (Hibiscus sabdariffa). Les prospections menées dans les principales zones de production de niébé au Niger, pendant quatre années consécutives (1982–1985) ont indiqué que les dommages de M. phaseolina se produisent dans tout le pays, excepté dans la région de Dosso dans la zone soudano - sahélienne. La présence de M. phaseolina dans la majorité des sols du Sénégal a été signalée depuis les années 60. Cependant, des pertes de production appréciables ne se sont produites qu’à partir de 1987. Depuis, des pertes élevées de rendement sont régulièrement observées sur le niébé, mais également parfois sur l’arachide dans la zone de Louga du Sénégal. Les rendements moyens en grain de niébé en champs paysans sont faibles (0,25 à 0,50 tonnes ha-1) et les pertes dues à la pourriture charbonneuse sont estimées à 10%. Ce qui correspond à une perte de 30.000 tonnes de niébé uniquement pour le Niger et le Sénégal pour une valeur estimée à $146 millions.

101 Résumé

M. phaseolina s’est avéré sensible aux fongicides et les traitements de semences et du sol peuvent réduire les infections et améliorer la germination. Cependant, la lutte chimique contre ce champignon est difficile, ni profitable et ni recommandée parce que le pathogène est transmis par le sol et par les semences. L’utilisation de cultivars de niébé résistants ou tolérants constitue les mesures de contrôle les plus appropriées contre M. phaseolina. Mais de tels cultivars ne sont pas encore disponibles. La solarisation, les amendements organiques, le maintien d’une bonne humidité du sol, la fumigation et l’utilisation des agents de lutte biologiques sont des méthodes potentielles de lutte contre les pathogènes telluriques. Cependant, il n’existe aucune méthode de lutte, qui à elle seule peut permettre de contrôler efficacement la pourriture charbonneuse. La pression de la maladie peut seulement être réduite si plusieurs mesures de contrôle préventives sont combinées dans une stratégie de gestion intégrée. Le chapitre 1 de cette étude qui en compte 6 fait le point de la connaissance sur la biologie et les méthodes de lutte contre M. phaseolina. Le chapitre 2 étudie les variabilités physiologique, génétique et pathogénique des isolats de M. phaseolina inféodés à trois systèmes de culture du niébé au Sahel. Pour cela, des isolats ont été obtenus de sols infestés et des tissus de plantes malades de niébé ou de mil provenant du Sénégal et du Niger. Les isolats ont ensuite été caractérisés pour leur potentiel de croissance à différentes températures, leur morphologie sur le milieu de culture PDA, leur profil génétique basé sur les séquences de la région ITS (internal transcribed spacer) et leur virulence sur trois cultivars de niébé. Un échantillon de 20 isolats choisi sur la base des caractéristiques de croissance sur PDA a été utilisé pour les études de pathogénicité sur des céréales. Le niébé cv. Mouride caractérisé par une sénescence retardée des feuilles (DLS) a été évalué pour la résistance dans un sol naturellement infesté par M. phaseolina. Les résultats ont montré que les isolats de M. phaseolina se comportaient différemment pour leur aptitude à croître à différentes températures, leur profil génétique, et leur pathogénicité sur les céréales. Les caractéristiques physiologiques et génétiques des isolats ne pas corrélées avec la pathogénicité. Cependant, nous avons montré pour la première fois, l’existence d’une certaine spécialisation pathogénique sur des céréales (excepté le fonio) des isolats de M. phaseolina obtenus de champs cultivés en mil et niébé. Les observations en champ et des études in vitro ont indiqué que le fonio n’est pas hôte et le mil un hôte peu sensible au M. phaseolina. C’est ainsi que l’influence de la monoculture du fonio (Digitaria exilis) et du mil (Pennisetum glaucum) sur la densité de l’inoculum du sol de M. phaseolina a été étudiée en condition naturelle. L’expérience a été conduite dans un champ naturellement infesté dans un dispositif en bloc aléatoire complet (RCDB). L’effet de trois ans de monoculture de fonio et de mil sur la survie de l’inoculum de Macrophomina a été évalué dans trois parcelles avec des

102 Résumé niveaux d’infestation de sol différents. A la quatrième année de culture, l’effet de ces pratiques sur le rendement du niébé a été également évalué (chapitre 3). Le fonio n’a pas été infecté par M. phaseolina alors que le système racinaire du mil hébergeait une faible densité de microsclérotes. Les rendements de niébé en fanes et en gousses avaient sensiblement augmenté après 3 ans de monoculture de fonio et de mil. Nous concluons que la rotation du niébé avec des graminées peut entraîner une diminution relativement rapide de la densité de l’inoculum du sol. Dans le cas d’une forte infestation, le fonio peut être cultivé pendant trois années successives pour réduire l’inoculum à un seuil non dommageable pour le niébé. Une expérience sur l’effet du compost sur l’inoculum du sol et la sévérité des symptômes de la pourriture charbonneuse (chapitre 4) a été conduite en champ. Les essais ont été installés dans des parcelles naturellement infestées. L’effet de 3 et 6 tonnes de compost ou de 6 tonnes de compost combiné à 50 kg d’engrais NPK ha-1 appliqué au trou de semis, sur le développement de la pourriture charbonneuse a été évalué pendant trois années consécutives. En outre, les effets combinés de 3 tonnes de compost ha-1 et de l’agent de lutte biologique Clonostachys rosea (108 CFU par g de compost) sur le développement de la pourriture charbonneuse et la production de niébé ont été étudiés. Nos résultats indiquent que dans les sols sableux du Sahel, un bon niveau de contrôle de la maladie et une augmentation substantielle des rendements du niébé peuvent être obtenus par l’amendement des sols avec 6 tonnes de compost ha-1. Une augmentation de rendement encore plus élevée est obtenue par amendement des sols avec 6 tonnes de compost combiné avec 50 kg de NPK ha-1 ou de 3 tonnes ha-1 de compost enrichi avec C. rosea. Le chapitre 5 traite des effets combinés de la solarisation et de l’amendement du sol avec des résidus de mil ou du contenu des panses sur la survie de M. phaseolina et le développement de la pourriture charbonneuse du niébé dans un sol naturellement infesté. Le dispositif expérimental était un plan en parcelle partagée ou split plot avec la solarisation comme facteur principal et les amendements comme facteur secondaire. La matière organique a été appliquée à la dose de 3 tonnes ha-1. Nos résultats ont montré qu’au niveau des parcelles amendées, la solarisation a élevé la température du sol jusqu’à 50°C pendant au moins 4 h jour-1 au cours du mois de juin, entraînant une réduction significative (44%) de l'inoculum du sol de M. phaseolina. L’amendement avec les contenus de panse (3 tonnes ha-1) a causé une réduction de 66% de l’inoculum initial dans les parcelles solarisées, alors que la solarisation n’entraînait pas une réduction significative des microsclérotes dans les parcelles amendées avec les résidus de mil. Ces observations montrent que dans les conditions où la solarisation seule ne permet pas un niveau de contrôle acceptable de la maladie, sa combinaison avec des amendements organiques réduisent significativement l’infection de M. phaseolina et

103 Résumé augmentent les rendements. Les amendements avec des matières organiques à teneur en N élevé, comme les contenus de panse utilisée dans cette étude se sont révélé les plus efficaces à contrôler la pourriture charbonneuse. La solarisation aussi bien que l’incorporation des résidus de mil ou de contenus de panse dans des sols humides peuvent doubler la production de niébé dans les sols pauvres et naturellement infestés de la zone aride et semi-aride du Sahel. En outre l’utilisation en agriculture des contenus de panses des abattoirs des grandes villes peut contribuer à la gestion durable des déchets urbains. En conclusion cette étude indique que les isolats de M. phaseolina inféodés à trois systèmes de culture du niébé dans le Sahel varient par rapport à la virulence et à la pathogénicité sur des cultivars de niébé et des espèces de céréales. Parmi les isolats de M. phaseolina le génotype (G9) très virulent sur le niébé est presque exclusivement présent au Sénégal et est inféodé au système monoculture de niébé. Pour la première fois nous avons trouvé des isolats de C. rosea dans le Sahel capables de contrôler efficacement M. phaseolina. En outre, l’étude a démontré qu’en incluant le fonio dans le schéma de rotation du niébé et du mil, en amendant les sols avec des résidus de mil ou le contenu des panses avec ou sans solarisation ou avec 3 tonnes ha-1 de compost enrichi avec C. rosea le développement de la pourriture charbonneuse peut être réduit significativement dans les parcelles infectées et les rendements de niébé améliorées en champ paysan. Les quatre méthodes de contrôle décrites dans cette étude pourraient être appliquées en combinaison de 2 à 4 traitements selon les conditions locales pour une gestion durable de la pourriture charbonneuse. Cette stratégie intégrée basée sur des traitements simples et à moindre coût pourrait être facilement adoptée par les paysans.

104 SAMENVATTING

Ogenboon (Vigna unguiculata (L.) Walp.) is het meest belangrijke voedingsgewas in de Sahel. Het wordt geteeld op een oppervlak van 12,5 miljoen hectare en is een waardevolle eiwitbron voor zowel mensen als dieren. Ongeveer 64% van de teelt van ogenboon is in west- en centraal-Afrika; de belangrijkste regio’s zijn in Nigeria (ca. 4 miljoen hectare en 1,7 miljoen ton opbrengst), Niger (3 miljoen hectare en 0,3 miljoen ton) en Senegal (0,12 miljoen ha en 0,08 miljoen ton). De belangrijkste limiterende factoren bij de teelt van ogenboon in de Sahel zijn gebrek aan water en voedingsstoffen. In Niger en Senegal wordt ogenboon vooral geteeld in de aride en semi-aride zone, waar de jaarlijkse neerslag beperkt is 250–500 mm. Macrophomina- rot, veroorzaakt door de schimmel Macrophomina phaseolina, is een belangrijke ziekte in ogenboon. Macrophomina-rot neemt de laatste decaden toe in verscheidene gewassen. Droogtestress, die maakt dat gewassen meer vatbaar voor infectie door M. phaseolina zijn, speelt wellicht een belangrijke rol. Sinds 1981 is Macrophomina-rot in toenemende mate in Niger waargenomen in sorghum (Sorghum vulgare), aardnoot (Arachis hypogea), okra (Hibiscus esculentus), sesam (Sesamum indicum), lablab (Dolichos lablab) en roselle (Hibiscus sabdariffa). Tijdens een inventariserend onderzoek in de jaren 1982–1985 bleek dat M. phaseolina behalve in het district Dosso wijd verbreid is in Niger. In Senegal komt Macrophomina-rot al voor sinds 1960, maar opmerkelijke opbrengstverliezen werden pas voor het eerst gemeld in 1987 in ogenboon en in aardnoot in Louga. De gemiddelde opbrengst in ogenboon is normaliter laag (0,21–0,50 ton/ha) en verliezen veroorzaakt door Macrophomina-rot worden in Niger en Senegal geschat op gemiddeld 10% (= 30.000 ton of $ 146 miljoen). Hoewel enige effectiviteit van fungiciden tegen M. phaseolina is aangetoond is de effectiviteit ervan onvoldoende door de talrijke aanwezigheid van in de bodem overlevende microsclerotiën. Bovendien hebben boeren niet de beschikking over fungiciden. Resistente of tolerante rassen zijn niet beschikbaar. Andere methoden ter beheersing van de ziekte zijn solarisatie, toediening van organische stof, handhaven van een hoger vochtgehalte in de grond, fumigatie en gebruikt van biologische bestrijding. Geen enkele van deze methoden blijkt de ziekte in voldoende mate te kunnen bestrijden. Daarom is dit proefschrift gericht op de toepassing van combinaties van diverse opties om te zien of een dergelijke geïntegreerde bestrijding wel effectief kan zijn.

105 Samenvatting

In hoofdstuk 2 is de variabiliteit in een reeks van isolaten van M. phaseolina uit Niger en Senegal onderzocht voor wat betreft de fysiologische, genetische en ziekteverwekkende eigenschappen van het pathogeen. Isolaten werden verzameld van grond en van geïnfecteerd plantenmateriaal afkomstig van drie teeltsystemen met ogenboon. Deze isolaten werden gekarakteriseerd met betrekking tot: groei bij diverse temperaturen, morfologie op aardappeldextroseagar, virulentie op drie cultivars van ogenboon en DNA-sequenties van de ITS-regio. Ook werd de mate van vorming van microsclerotiën van 20 isolaten onderzocht in verscheidene gewassen. Tot slot werd de resistentie van cv. Mouride (die ten opzichte van andere isolaten langzaam veroudert) tegen een reeks van isolaten van M. phaseolina onderzocht. De isolaten verschilden slechts in beperkte mate in temperatuurgevoeligheid, ITS-sequentie en pathogeniteit op granen. De groepen die onderscheiden konden worden op basis van fysiologische of genetische eigenschappen stemden niet overeen met de groepen die onderscheiden konden worden op basis van pathogeniteit. Wel werd, voor de eerste keer, enige specialisatie in pathogeniteit waargenomen ten aanzien van granen (behalve fonio) voor isolaten die waren verkregen van velden met gierst na ogenboon. Veldwaarnemingen en experimenten gaven aan dat fonio niet een waardplant is van M. phaseolina, en gierst een zwakke waardplant. Daarom werd het effect van de teelt van fonio (Digitaria exilis) en gierst (Pennisetum glaucum) op de bodembesmetting door M. phaseolina in een natuurlijk besmet veld onderzocht (hoofdstuk 3). Fonio bleek niet te worden geïnfecteerd door M. phaseolina en alleen de wortelsystemen van gierst hadden geringe hoeveelheden microsclerotiën. De opbrengst van ogenboon was significant hoger na een driejarige teelt van fonio dan na een driejarige teelt van gierst. Vruchtwisseling met een grasachtig gewas leidt tot een relatief snelle afname van de bodembesmetting door M. phaseolina. Bij een zware bodembesmetting is een driejarige teelt met fonio noodzakelijk om de teelt van ogenboon weer mogelijk te maken. Het effect van compost op Macrophomina-rot in ogenboon werd onderzocht in een van nature besmet veld (hoofdstuk 4). De behandeling bestond uit de toediening van compost aan plantgaten. Er waren de volgende behandelingen: 3 ton compost per hectare, 6 ton compost per hectare, 6 ton compost per hectare aangevuld met 50 kg kunstmest (NPK) per hectare en 3 ton compost per hectare aangevuld met de biologische bestrijder Clonostachys rosea (108 kolonievormende eenheden per gram compost). Het bleek dat 6 ton compost per hectare een voldoende bestrijding van Macrophomina-rot in ogenboon te zien gaf. Zowel aanvulling van kunstmest of de behandeling van 3 ton compost aangevuld met C. rosea gaven nog iets betere resultaten te zien.

106 Samenvatting

In hoofdstuk 5 werd het effect van de gecombineerde toepassing van solarisatie van de grond en toediening (3 ton per hectare) van gewasresten van gierst of pensafval van slachterijen onderzocht op de overleving van M. phaseolina in de grond en op de ontwikkeling van Macrophomina-rot in ogenboon in een van nature besmette veldgrond. Solarisatie in juni had een temperatuurverhoging van de bovenste laag van de grond tot gevolg die leidde tot temperaturen van ten minste 50°C gedurende 4 dagen of langer. Dit leidde tot een afname van de bodembesmetting van M. phaseolina met 44%. Pensafval leidde zelfs tot een aanvullende reductie met 66%, terwijl gewasresten van gierst geen aanvullend effect hadden op de solarisatie. Het blijkt dus dat de effecten van solarisatie sterk verbeterd kunnen worden door toediening van een actieve organische stof. Alle behandelingen, inclusief de toediening van gewasresten van gierst, leidden tot significant hogere opbrengsten van ogenboon. De resultaten van deze studie kunnen leiden tot een nuttig gebruik van pensaval. Deze studie geeft aan dat isolaten van M. phaseolina afkomstig van verschillende teeltsystemen verschillen in virulentie en vermogen tot infectie van gewassen en cultivars die niet zeer vatbaar zijn voor dit pathogeen. Eén genotype was specifiek gelieerd aan continue ogenboonteeltsystemen in Senegal. Voor het eerst zijn er lokaal verkregen isolaten van Clonostachys rosea effectief gebleken tegen M. phaseolina. Tot slot zijn er duidelijke opties gecreëerd voor de geïntegreerde beheersing van Macrophomina-rot: vruchtwisseling met fonio, solarisatie in combinatie met toediening van pensafval en biologische bestrijding met C. rosea.

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LIST OF PUBLICATIONS

Ndiaye, M., Bashir, M., Keller, K. and Hampton, A. O. 1993. Cowpea viruses in Senegal, West Africa: Identification, distribution, seed transmission, and sources of genetic resistance. Plant Dis. 77: 999–1003. Ndiaye, M. and Dollet, M. 1996. La maladie du rabougrissement de l'arachide. Caractérisation symptomatologique et sérologique de nouveaux isolats du peanut clump virus (PCV). Parasitica 51: 131–142. Cissé, N., Ndiaye, M., Thiaw, S., and Hall, A. E. 1995. Registration of ‘Mouride’ cowpea Crop Science 35: 1215–1216. Cissé, N., Ndiaye, M., Thiaw, S. and Hall, A. E. 1997. Registration of 'Melakh' cowpea. Crop Science 37: 1978. Cisse, N., Ndiaye M. and Sène A. 2005. Registration of 'Yacine' cowpea. Crop Science 45: 413–414. Ndiaye, M., Termorshuizen, A. J. and van Bruggen A. H. C. 2006. Effect of rotation of cowpea (Vigna unguiculata) with Digitaria exilis and Pennisetum glaucum on Macrophomina phaseolina densities and cowpea yield. Submitted to Crop Protection. Ndiaye, M., Termorshuizen, A. J. and van Bruggen A. H. C. 2006. Effects of compost amendment on charcoal rot (Macrophomina phaseolina) development of cowpea. Submitted to Crop Protection. Ndiaye, M., Termorshuizen, A. J. and van Bruggen A. H. C. 2006. Combined effects of solarization and organic amendment on charcoal rot caused by Macrophomina phaseolina in the Sahel. Accepted for publication in Phytoparasitica. Ndiaye, M., Termorshuizen, A. J., van Bruggen A. H. C., Groenewald, J. Z. and Crous, P. W. 2007. Physiological, genetic, and pathogenic variability in Macrophomina phaseolina, the causal agent of charcoal rot. To be submitted.

109

CURRICULUM VITAE

Mbaye NDIAYE was born in 1960 in Senegal. From 1980-1986, he was registered in Vacil Kolarov Agricultural Institute, Plovdiv, Bulgaria, where he graduated with an MSc degree in crop protection. After completing another MSc in agricultural sciences in 1989 at the Catholic University of Louvain-Neuve, Belgium, he joined the Senegalese Agricultural Research Institute. He was the officer in charge of the Legume Pathology Department at the National Agricultural Research Center, Bambey, Senegal, from Mai, 1990 to September 1995. During this period, he attended many workshops and symposia and practical training courses at IITA, Ibadan, Nigeria; OSU, Corvallis, USA; CIRAD, Montpellier, France. His area of research was mainly breeding of cowpea for resistance to viruses, bacterial blight and charcoal rot. Since October 1995, he has been working as lecturer in mycology and bacteriology at the Regional Center AGRHYMET, Niamey, Niger. Besides his teaching activities, he conducts research on control methods for Macrophomina phaseolina, which resulted in this PhD thesis at Wageningen University. He visited Wageningen University three times, for 2 weeks, 3 months and 3 months in 1998, 2006 and 2007, respectively, to write a research proposal, analyse research data, write the PhD thesis and take post- graduate courses.

111

PE&RC PhD Education Certificate

With the educational activities listed below the PhD candidate has complied with the educational requirements set by the C.T. de Wit Graduate School for Production Ecology and Resource Conservation (PE&RC) which comprises of a minimum total of 32 ECTS (= 22 weeks of activities).

Review of Literature (6 credits) - Integrated perspectives on charcoal rot caused by Macrophomina in cowpea in the Sahel. The document was discussed at the PE&RC discussion group chair by prof. van Bruggen as well as in AGHRYMET (1999) Writing of Project proposal (7 credits) - Ecology and management of charcoal rot caused by Macrophomina phaseolina on cowpea in the Sahel Post-Graduate Courses (7.4 credits) - The use of Computers in Microbiology for Data Retrieval and Information Management; MSDN, Dakar Senegal (1994) - Conception and statistical analyses of agronomic trials; ISRA, Dakar Senegal (1995) - Biological control of pest and of tropical crops; IIITA/UNB/NATURA/NECTAR, Cotonou Benin (1996) - Multivariate analysis; WUR, Wageningen the Netherlands (2007) Deficiency, Refresh, Brush-up and General courses (2.5 credits) - Integrated disease management of bacterial diseases and root and stem rots of cassava and cowpea; International Institute of Tropical Agriculture, Cotonou Benin (1999) - Molecular and Evolutionary Ecology; WUR the Netherlands (2007) Competence Strengthening / Skills courses (1.3 credits) - Conception, development and utilization of a lecture online; AUF/Université Abdou Moumouni, Nianey Niger (2006) - Techniques for Writing and Presenting a Scientific Paper; WUR the Netherlands (2006)

113 Name of the discussion group, local seminars and other scientific meetings (5.2 credits) - Agricultural production systems in the temperate regions/ Ariena van Bruggen (1999-2006) - Harmonization of seed regulations in the CILSS countries: quality control and phytosanitary standards (2003) - Legal texts an mechanisms of operation of the Sahelian committee of the conventional and transgenic seeds (2005) - Scientific and pedagogic committee meeting (2006) PE&RC Annual Meetings, Seminars and Introduction Days (2.1 credits) - Seminar on renewable energy (2002) - PE&RC Day ,The scientific agenda: ”who pulls the strings”(2006) - Aid making decision tools in integrated crop protection- Diagnostic Advisory Rule-based Expert System for Integrated Pest Management in Solanaceous Crop Systems (2006) International Symposia, Workshops and Conferences (4.2 credits) - 2nd World Cowpea Conference, CRSP-IITA, Accra, Ghana; Poster presentation: Characterization of a new potyvirus in Senegal (1995) - 2nd International conference of Virology and Mycology, Federation of African Virology and Mycology-Cameroon, Yaoundé: Oral presentation; Effects of cropping systems on charcoal rot development of cowpea (1999) - Darwinian agriculture: the evolutionary ecology of agriculture symbiosis (2007) Laboratory Training and Working Visits (4.3 credits) - Serological techniques for diagnostic of cowpea viruses; Oregon state University, Corvallis USA (1992) - Diagnostic methods of peanut viruses; CIRAD-CP, Montpellier, France (1994) - Lab & greenhouse visits and discussion on use of mychorrizal fungi in plant protection; Danish Institute of Agriculture Sciences, Denmark (2005) Courses in which the PhD candidate has worked as a teacher - Introduction to plant protection; DFR/TSPV, 7 days - General Mycology; DFR/TSPV, 14 days - Epidemiology of Plant pathogens; DFR/IPV - Seed production and protection; DFR/IPV Supervision of MSc student(s) - Plant pathology; 15 days per student, 50 MSc students

114