Incidence and severity of Fusarium wilt on Bambara nut (Vigna subterranea L.) landraces in Western Kenya

Bambara nut, Vigna subterranea (L.) Verdc. is one of the indigenous legume crops grown in Kenya. The crop is highly nutritious and drought tolerant with the ability to produce higher yields than other legumes such as common groundnuts and beans produced under the same conditions. Bambara nut demand is on the increase due to the current focus on neglected underutilized crop species. The crop yield is however low because of biotic stresses among others. Fusarium oxysporum Schlechtend f.sp. voandzeia is one of the most destructive fungal pathogens affecting Bambara nut in Kenya. A greenhouse experiment was therefore carried out to determine the incidence and severity of the disease on local landraces of Bambara nut. The experiment was laid out in a completely randomized design replicated three times. Disease incidence and severity varied significantly with landrace. The maroon speckled and brown dark eyed landraces had the highest disease incidences (80.5% and 80.0%). Followed by brown light eyed, maroon and black landraces with diseases incidences of 79.5%, 78.9% and 78.6% respectively and lastly the red landrace with least disease incidence of 76.2%. Disease severity also varied with landrace. The maroon followed by the brown dark eyed landraces had the highest disease severities of 45.5% and 44.3% then the red, maroon speckled and black landraces with severities of 43.5%, 43.1% and 42.8% respectively. The Brown light eyed landrace had the least disease severity of 42.6%. There was a significant interaction between landrace and days after inoculation with respect to disease severity. At 75 days after inoculation disease severity was similar for all the landraces with approximately 40% severity. The area under disease progress curves (AUDPC) varied with landrace. The Maroon landrace had the highest AUDPC while the Brown landrace had the least area. The study confirmed the virulence of the pathogen on the crop and the need to manage the disease for improved yield performances.


Introduction
Bambara nut, Vigna subterranea (L.) Verdc. is one of the indigenous legume crops grown in sub Saharan Africa. The crop ranks third after ground nuts (Arachis hypogaea L.) and cowpeas (Vigna unguiculata L.) in most parts of Africa (Mkandawire, 2007). Bambara nut seed is highly nutritious containing about 49-63.5% carbohydrates, 15-25% protein, 4.5-7.4% lipids and 5.2-6.4% fiber (Bamshaiye et al., 2011). The crop is drought tolerant with the ability to produce higher yields than other legumes such as groundnuts and beans produced under the same conditions (Masindeni, 2006). Bambara nut is important in low input agricultural production systems because of its ability to fix atmospheric nitrogen into the soil with the aid of Bradyrhizobium bacteria. The demand for the crop is rising because of the added effects of its medicinal value (Directorate of Plant Production, 2011).
In Kenya, Bambara nut is mainly grown at the Coast, Eastern, Western and Nyanza regions (Wasula et al., 2012;and Wasula, 2014). The crop's production has however been low because of diseases among other factors. Fusarium wilt caused by F. oxysporum Schlechtend f.sp. voandzeia is among the most important diseases of Bambara nuts in Kenya (Cook, 1978). The disease is prevalent in most Bambara nut growing regions (Begemann, 1986). Fusarium oxysporum attacks Bambara nut at all stages of development. The propagules gain entry into the plant through cut surfaces of seeds, damaged roots and stem tissues of young and stressed plants, infected seeds and through wounds caused by insects .
Generally the recommended control practices for F. oxysporum include use of resistant varieties, fungicidal seed treatments, adjustment of planting dates, use of biofumigants, bioagents and crop rotation techniques among others (Blanca et al., 2004;Ajiloba and Babalola 2013;and Kemal et al., 2015). In Kenya, farmers grow the crop during the short rainy season when the disease pressure is low. There is an increasing demand for Bambara nut arising from several alternative uses such as medicinal values, need for healthy diets and its adaptability to unpredictable climate changes. The climate change implies that farmers have to be prepared for different conditions that may affect disease incidence and severity and therefore need to plant Fusarium wilt tolerant landraces. Host plant resistance is one of the most important options for disease control but little work has been done to identify and quantify its status among Bambara nut landraces.
An experiment was carried out to determine the incidence and severity of Fusarium wilt on Bambara nut in Western Kenya with an aim of identifying the tolerant landraces.

Site description
Bambara nut samples were collected from farmers' fields in Busia county which lies between latitudes 0°30' and 0°45' N and longitudes 33º55' and 34°25' E, at an altitude of 1200 m above sea level. The area experiences a mean annual rainfall of 900 mm-1800 mm and annual mean temperature of between 17 °C and 22 °C. The area lies within the Lower Midland 1 and 2 agro ecological zones with predominantly humic acrisol soils (Jaetzold et al., 2006).

Sampling and fungal isolation
Bambara nut plants showing symptoms of the disease were collected from farmers' fields in Busia County and transported in press seal plastic bags (75 mm  100 mm) in a cool box to Egerton University's Biological Sciences laboratory for pathogen isolation and identification. The Kristin and James (2000) protocol was used for fungal isolation. Root tissues were washed in running tap water, cut into 1 cm portions, surface sterilized in 1.5% NaOCl for one minute, double rinsed in sterile distilled water and blot dried between sterile paper towels.
Glass Petri dishes (Pyrex®) sterilized in the oven at 160 °C for 45 min were used to grow the isolates. The sterilized samples were aseptically plated on Czapekdox agar medium (Sodium nitrate 3 g, Potassium chloride 0.5 g, Di-potassium hydrogen phosphate 1 g, Sucrose 30 g, Magnesium sulphate 1 g, Ferrous sulphate 0.01 g, Agar 15 g and water 1 liter), a selective media for Fusarium growth.
The medium was autoclaved at 121 °C for 20 min allowed to cool down to touch temperatures before being amended with 5 ml/liter of streptomycin sulphate to eliminate bacteria contaminants. The sterile plates containing the samples were sealed using parafilm and incubated at 25-26 ºC, before examination for colony formation within 2-14 days (Maina et al., 2015). The Fusarium wilt fungus was purified by sub-culturing on Potato Dextrose Agar (PDA) media and identified with the help of relevant literature .

Pathogenicity test
The in vivo assay was carried out in the greenhouse at Egerton University based on the Ros et al. (2005) method. A pure sub culture was made from cultures isolated from infected Bambara nut roots and an inoculum prepared from these cultures. Sterilized PDA plates amended with 5 ml/liter of streptomycin sulphate were inoculated with three mycelia plugs of Fusarium oxysporum from actively growing regions of the mycelial growth and incubated for 7 days at 25-26 ºC. The spores from 7-day old cultures were scrapped off from the petri dishes surfaces by adding sterile distilled water so as to obtain a suspension that was filtered through one layer of miracloth and the concentration adjusted using a haemocytometer to 10 6 conidia/ml. Seeds of the black and red landraces of Bambara nut were surface sterilized in 1% sodium hypochlorite for three minutes, rinsed in three changes of distilled sterile water and air dried in the laminar flow. Three seeds per landrace were then planted in pots (18 cm long and 19 cm wide) containing 2.5 kg of sterilized sandy loam soil (autoclaved at 120 °C for 1 h for three consecutive days) with adequate watering. At 14 days after emergence 10 ml of a 10 6 conidia/ml of spore suspension was applied over the base of the hypocotyl of the seedlings using a 10 ml syringe. Development of pathogenicity signs and symptoms was observed on a weekly basis and records taken. Re-isolation was then carried out to confirm isolates identity 12 weeks after planting.

Disease incidence and severity
The experiment was laid out as a completely randomized design replicated three times in a greenhouse. The most cultivated Bambara nut landraces in Busia county (black, red, maroon, maroon speckled, brown light eyed and brown dark eyed) were used in the study. Two seeds per landrace were germinated in pots (measuring 18 cm in length and 19 cm in diameter) containing 2.5 kg of sterilized sandy loam soil (autoclaved at 120 °C for 1 h for three consecutive days) with adequate watering. Fourteen day old seedlings were then inoculated with 10 ml of a 10 6 conidia/ml of spore suspension of F. oxysporum at the base of the hypocotyl using a 10 ml syringe as described by Kristin and James (2000).

Disease assessment
Wilt incidence and severity were estimated at 10-day intervals using a scale of 0-5 based on leaf yellowing rating as described by Abdou et al. (2001). The rating included; 0 = Healthy 1 = One leaf yellowing 2 = More than one leaf yellowing 3 = One wilted leaf 4 = More than one leaf wilted 5 = Completely dead plants Disease Severity Index (DSI) was determined using the formula described by Liu et al. (1995)  Disease Incidence (DI) at 45 days after inoculation was determined using the equation proposed by Cooke (2006) for a period of 60 days.

DI = (No. of infected plant units/total no. of plant units assessed)  100
The area under disease progress curve (AUDPC) which is a quantitative summary of disease intensity over time for each landrace was calculated using the formula suggested by Pandy et al. (1989).

Colony characteristics
The mycelia were white cottony to pink with the aerial part becoming tinged with purple coloration (Plate 1). The reverse was non-descript pale to yellow (Plate 2). Microconidia were abundantly born on phialides with straight, curved or ellipsoidal shapes. The macroconidia were sparsely found accumulating along the hyphae or scattered. They were often 3-5 septate and pointed at both ends, more ellipsoidal than microconidia (Plate 3). Chlamydospores which developed after seven days (i.e., about 14 days of incubation) were either smooth or rough walled, abundant and formed terminally or intercalary (Plate 4).

Pathogenicity
Fusarium wilt symptoms occurred 45 days after inoculation (Plate 5). The first indication of the disease was yellowing of leaves that intensified with time, before the entire crop eventually wilting. Destructive sampling showed brown vascular discolorations. The isolate was highly virulent with all the two landraces (black and red) being susceptible to the pathogen. Fusarium oxysporum identical to the parent isolate was re-isolated from the diseased root tissues.

Incidence and severity of Fusarium wilt
The landraces had high and varied disease incidence and severity (Table 1). The maroon speckled and brown dark eyed landraces had the highest disease incidences of 80.5% and 80.0%. Followed by brown light eyed, maroon and black landraces with incidences of 79.5%, 78.9% and 78.6% respectively. The red landrace had the least disease incidence of 76.2%.
Disease severity varied with landrace. The maroon and brown dark eyed landraces had the highest disease severities of 45.5% and 44.3%. Followed by the red, maroon speckled and black landraces with severities of 43.5%, 43.1% and 42.8% respectively. The brown light eyed landrace had the least disease severity of 42.6%.

Area under disease progress curves (AUDPC)
The AUDPC for severity were generated for all the landraces and at 75 days after inoculation all the landraces had similar disease severity of approximately 40% (Figure 1). Incidence and severity varied significantly with landrace as well as days after inoculation (Table 2). Interaction between landrace and days after inoculation was significant for the disease severity. The AUDPC varied with landrace ( Figure 2). The maroon and brown dark eyed landraces had the highest AUDPC, followed by the red, maroon speckled and black landraces respectively. The brown light eyed landrace had the least AUDPC.

Discussion
Fusarium wilt of Bambara nuts is among the most destructive soil-borne diseases of the crop. The disease is prevalent in most Bambara nut growing regions and it attacks Bambara nut at all stages of development. Fusarium wilt was isolated in most of the samples collected from farmers' fields in Busia. Wakelin et al. (2008) similarly reported abundance of Fusarium species in cultivated soils and mostly associated with plant roots. The colony characteristics are consistent with the descriptions by Kleczewski and Egel (2011). Pathogenicity tests proved presence of the disease since the symptoms expressed in the greenhouse were similar to those observed in farmers' fields. Ebbels and Billington (1972) in Tanzania similarly reported presence of Fusarium wilt on Bambara nut landraces. Occurrence of symptoms varied with landraces ranging from 14 days to 50 days after inoculation. The characteristic symptoms which included yellowing of leaves, wilting of the plant and brown vascular discoloration throughout the trial period were in complete compliance with those observed by Sharma (2011).
In the incidence and severity studies, the symptoms were seen starting from the vegetative phase through flowering with varying degrees among the landraces. Zemouli-Benfreha et al. (2013) and Landa et al. (2004) similarly reported presence of Fusarium wilt symptoms in all stages of chickpea development. This is probably because the pathogen is soil borne (Zhao et al., 2014) hence infects the plant starting from seedling phase. However at 75 days after inoculation all the landraces had similar degree of disease severity of approximately 40%. It is around this period that the crop is flowering and initiating pod set and also the inoculum levels are high due to rapid sporulation by the pathogen within the vascular system. Chaudhry et al. (2006) similarly observed high Fusarium wilt intensity at flowering and podding stages of chickpea development.
The high disease incidence and severity on the selected landraces is an indication of the virulence of the pathogen on the crop. Of all the tested landraces none proved to be tolerant to the disease implying that there is no resistant type. Cook (1978) also reported Fusarium wilt in Bambara nut growing areas in Kenya. There was a variation in the manner in which the landraces performed in relation to incidence and severity. Maroon speckled landrace recorded the highest disease incidence but it was not the worst hit in terms of severity. Instead the maroon landrace had the highest disease severity while the brown landrace had the least severity. This shows that genetically the brown landrace is more tolerant to the disease than the other landrace. Aslam et al. (2013) similarly noted influence of genetic makeup of different chickpea cultivars on the Fusarium wilt severity.

Conclusion
Isolation of Fusarium wilt from most of the samples collected from farmers' fields in Busia indicates presence of the disease in the fields. The high incidence and severity on the selected local landraces calls for efforts of plant breeders to work towards developing Bambara nut varieties that are resistant to the Fusarium wilt so as to boost the crop's yield performance.