Differential reaction of cowpea genotypes to brown blotch disease (Colletotrichum capsici) in Burkina Faso

1 Laboratory of Plant Pathology, Institut de l’Environnement et de Recherches Agricoles (INERA), 01 BP 476 Ouagadougou, Burkina Faso. 2 LMI Patho Bios (INERA-IRD), Ouagadougou, Burkina Faso. 3 Laboratory of Genetic and Plant Biotechnology, Institut de l’Environnement et de Recherches Agricoles (INERA), 01 BP 476, Ouagadougou 01, Burkina Faso. 4 Laboratory of Biosciences/Phytopathologie, Université Ouaga I Pr. Joseph KI-ZERBO, Burkina Faso. 5 Laboratory of Biosciences/Genetic and Biotechnology, Université Ouaga I Pr. Joseph KI-ZERBO, Burkina Faso.


INTRODUCTION
Cowpea (Vigna unguiculata (L.) Walp.) is an important grain legume crop in Sub-Saharan Africa (Adegbite and Amusa, 2008).The crop is grown worldwide with an estimated cultivation area of about 12 million hectares annually with the production of over 10 million metric tons a year (FAOSTAT, 2011).About 65 to 70% of the world's cowpea grain production occurs in the west and the central part of Sub-Saharan Africa.Cowpea provides *Corresponding author.E-mail: gilthiol@yahoo.fr.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License food and nutrition for humans and feed for livestock.However, in Burkina Faso and other African countries, cowpea production is subject to many biotic constraints such as soil-borne and seed-borne fungal pathogens including Colletotrichum capsici, the causal agent of brown blotch disease, which can result in yield losses of between 42 and 100% in tropical agriculture (Sereme, 1999;Banerjee et al., 2007;Adegbite and Amusa, 2008;Torres-Calzada et al., 2011).C. capsici is a major constraint to many crop production causing severe losses both in pre-and postharvest decay (N'Guettia et al., 2013;Saxena et al., 2014;Chandra et al., 2009).
Infection by C. capsici in cowpea is particularly devastating due to its hemibiotrophic nature (Hyde et al., 2009).Additionally, the pathogen occurs in different races.For instance, Emechebe (1986) identified eight races of C. capsici associated with brown blotch disease in Nigeria; four out of the eight races were specific to Guinea and Sudan Savanna while the remaining four were to the rainforest area.Sereme (1999) reported 12 pathogenic groups of Colletotrichum spp.including C. capsici associated with brown blotch disease in Burkina Faso.Based on molecular characterization with a specific primer pair, Thio et al. (2016) identified four variants of C. capsici in Burkina Faso.
Several C. capsici management techniques including cultural control, use of plant extracts (Sereme, 1999;Mark et al., 2015;Mark and Channya, 2016), use of chemicals, biological control (Chacko and Gokulapalan, 2015) and use of tolerant and resistant cultivars (Adebitan et al., 1992;Amusa et al., 1994) have been recommended.However, no single specific management program can eliminate brown blotch disease in cowpea (Obi and Barrusa-Vargas, 2014).Breeding for resistance is also the most economical for growers as well.
In this study, spraying method was used to study the pathogenicity and resistance of cowpea genotypes to C. capsici strains originating from different locations in Burkina Faso.

Collection of isolates of Colletotrichum spp.
Cowpea plant parts (leaves, stems, pods) naturally infected by Colletotrichum spp.were collected from farmers' fields in three agro-ecological zones of Burkina Faso during the period from October to November 2013.The locations included Saria, Kamboinse and Kouaré (North Soudanian zone), Farako Bâ and Gaoua (Soudanian zone), and Pobe Mengao (Sahelian zone).The cowpea plant materials were surface-sterilized in 70% (v/v) ethanol for 1 min followed by immersion in sodium hypochlorite (NaOCl) 1% (v/v) for 5 min and three successive rinses in sterilized distilled water.After disinfection, the materials were allowed to dry under laminar flow hoods for 15 min.Tissues of approximately 4 mm 2 in size were placed in Petri dishes containing moistened blotting paper.Plates were incubated in alternating cycles of light/darkness (12 h/12 h) for 7 to 9 days at 28°C.The Colletotrichum species were identified based on the growth habit characteristic and morphology of acervuli and conidia and on available identification keys (Marthur and Kongsdal, 2003).
Sporulating cultures of C. capsici were aseptically transferred in Petri dishes containing potato dextrose agar (PDA), which was previously autoclaved for 15 min at 121°C and then supplemented with streptomycin (0.3 µg/l of PDA) to prevent bacterial contamination.The Petri dishes were incubated at 24°C under UV light in alternating cycles of light/darkness (12 h/12 h) for 7 days.

Single spore production
A pure single spore culture was obtained from each C. capsici isolate after 7 days of growth on PDA. 100 ml of distilled water were added to the fresh fungal culture, and 200 μL of the suspension was spread on PDA media culture and incubated for 12 to 24 h depending on cell germination and growth.Three to five single cells or mycelium from each isolate were transferred to new Petri dishes containing PDA.After 7 days of growth, pure cultures of single spore were stored at 20°C before use.

Pathogenicity tests
A total of 36 single spores C. capsici (Table 1) were used to carry out pathogenicity tests in greenhouse conditions.These isolates were used for artificial inoculation of three differential cowpea genotypes namely KVx61-1, KVx396-4-5-2-D and "Moussa Local".The response of these three cowpea lines have been identified by Sereme (1999).Cowpea seeds were sterilized in 1% sodium hypochlorite (NaOCl) for 5 min, rinsed three times successively with distilled water, and dried on laminar flow hoods for 24 h.Fifteen to twenty seeds were sown in duplicates in 2.5 L pots containing sterilized soil mixture of sand and forest soil in a 1:2 ratios (v/v).Fourteen days after sowing (DAS), cowpea plants were inoculated (the whole surface of plants) with a concentration of approximately 10 6 spores/ml of each Colletotrichum isolate.To favor spores penetration inside the plant, the inoculated plants were maintained at 22 ± 1°C in humid (RH > 78%) conditions for 5 days.Then, the cowpea plants were examined for brown blotch disease symptoms after 7, 14 and 21 days after inoculation (DAI).
The disease severity index was based on a 1 to 5 scale, where 1 = no symptoms, 2 = small spots of brown blotch in the stem, 3 = coalescent spots on the stem, 4 = coalescent spot with presence of acervuli but surviving plant, 5 = withered stem and plant death (Figure 1) (Sereme, 1999), and on the formula proposed by Allen et al. (1981) as follows: Where: Xi = the note of disease for each plant; ni = individual number of category Xi; N = total number of observed plant; E (Xi) = scale range.
Analysis of variance (ANOVA) was performed using Gen Stat 12 th edition on disease incidence and severity data.All the data for disease severity were subjected to Arc sine transformation before

Seedling resistance evaluation of brown blotch disease
Forty one cowpea lines were screened for their resistance or susceptibility to brown blotch disease under greenhouse conditions.The seeds were previously surface sterilized in sodium hypochlorite (NaOCl) 1% (v/v) for 5 min and air dried for 24 h before sowing.A mixture of sand and field soil (v/v=1:2) was autoclaved at 121°C for 1 h and used to fill up 75% of the 10 L pot .Two replicates were set up for each cowpea accession and for each C. capsici isolates in a split plot design.The most virulent strain of C. capsici from each Agro-ecological zone of the country, designated Ccap-PO, Ccap-FA and Ccap-SA were used for plant inoculation (Figure 2).About 15 to 20 seeds were sowed in each pot for plant growth.The spore suspension from fresh fungi culture of 7 days was prepared and adjusted to a concentration of 10 6 spores/ml.The C. capsici suspension was applied by spraying over the entire surface of the individual plant and plant incubated for symptoms notation.Disease incidence and severity were determined using the same methods described for the pathogenicity test.Seedlings were also scored for disease severity using a modified scale (Sereme, 1999) as shown in Table 2.   pathogenic groups or races of C. capsici were identified based on their virulence or aggressiveness.Therefore, Agglomerative Hierarchical Clustering (AHC) analysis was used to predict 4 classes of pathogens in terms of disease severity at 75% of Pearson correlation coefficient (Figure 3).This analysis also indicated a high similarity coefficient within class at 84 (49%) against 15 (51%) for between classes.Thus, the Classes 1 and 2 contain the most pathogenic strains of C. capsici.All of the isolates of Saria were identified as the most virulent and are associated with the cowpea genotype KVX396-4-5-2D (Tables 3 and 4).Among these isolates, the strain 096-SA-2 was the most devastating with 78, 82 and 10% of severity, respectively, for KVX61-1, KVX396-4-5-2D and Moussa Local (Table 3).Isolates 096-SA-2, 079-PM-2 and 071-FA-6 were the most aggressive for the North Soudanian, Sahelian and Soudanian agro-ecological zone, respectively.Disease severity and incidence varied across the three agro-ecological zones.These three isolates are newly designated as Ccap-SA (096-SA-2), Ccap-PO (079-PM-2) and Ccap-FA (071-FA-6).The analysis of disease incidence and severity due to the virulent pathogens showed a highly significant difference (P < 0.001) among the isolates, genotypes and their interactions (Table 5).Among the three cowpea lines used for the test, KVX61-1 (28% severity) was the most susceptible followed by KVX396-4-5-2D (18%) while Moussa Local (7%) seemed to be resistant.Sereme (1999) reported that the most pathogenic groups of Colletotrichum spp.associated with brown blotch disease of cowpea were from Soudanian zone and were collected on the site of Farako Ba.The results of this study indicate a progression of C. capsici virulence around the country.The most pathogenic strains belonged to the C. capsici variant 1 which were identified by using internal transcribed spacer (ITS) phylomarkers in a previous study (Thio et al., 2016).The strains 035-GA-5 and 020-KB-5 presented the lowest values of aggressiveness and correspond, respectively to variants 2 and 4 of C. capsici.

A specific reaction of cowpea genotypes to C. capsici isolates
The screening of 41 cowpea cultivars in greenhouse condition involves identifying potential source of cowpea resistance.The results indicated highly significant difference (P < 0.05) in disease severity among cultivars, isolate effects and their interactions (Table 6).These results indicate high genetic variability in cowpea cultivars to brown blotch disease.All cowpea cultivars present approximately the same incidence at 14 DAI which is not significant.The observations also showed that the cowpea cultivars were more severely affected 21 days after inoculation than 14 DAI.The current study indicated  that 76% of cultivars tested were susceptible to brown blotch disease.The results revealed that among the 41 cowpea lines screened, the cultivar KN-1 showed a specific resistance to the three pathotypes.It was consistently brown blotch disease resistant and should be a good source of resistance genes for susceptible cowpea lines.

Figure 1 .
Figure 1. Brown blotch disease annotation symptoms on 1 to 5 scale.

Figure 2 .
Figure 2. Colony surface of strains: a: Ccap-PO; b: Ccap-FA and c: Ccap-SA, in PDA media, after 7 days of growth.
the same letter in a column are not significantly different at P ≤ 0.05.***Most pathogenic strain from agro-ecological zone.

c
Means followed by the same letter in a column are not significantly different at P ≤ 0.05 *** or ** or *= most pathogenic strain from agro-ecological zone; R= Resistant, S= Susceptible.

Table 3 .
C. capsici strains virulence associated to brown blotch disease on three cowpea genotypes at 14 days after inoculation.

Table 4 .
C. capsici strains virulence associated to brown blotch disease on three cowpea genotypes at 21 days after inoculation.

Table 5 .
ANOVA of mean disease incidence in cowpea at 14 days after inoculation due to C. capsici virulence.

Table 6 .
ANOVA of genetic variability of cowpea cultivars in brown blotch disease severity at 21 days after inoculation.
**Highly significant difference; CV = Coefficient of variation; df = Degree of freedom; Pr = Probability of F value.

Table 7 .
Differential reaction of 41 cowpea cultivars at 14 and 21 days after inoculation in C. capsici pathotypes.