Identification of Fusarium Species Associated with Onion ( Allium cepa L.) Plants in Field in Burkina Faso

Many fungi limit onion production in Burkina Faso. This study aims to identify the main Fusarium species associated with onion plant in field in order to determine those involved in seedling damping-off and bulb rot, and develop adequate management strategies of these diseases. For this purpose, 36 isolates of Fusarium were isolated from onion plants in 17 sites and subjected to molecular analysis and biometric characterization. The results revealed that the isolates belong to five Fusarium species: Fusarium oxysporum (44.44% of the isolates), Fusarium proliferatum (41.66%), Fusarium solani (5.55%), Fusarium fujikuroi (5.55%) and Fusarium thapsinum (2.77%). Fusarium oxysporum, F. proliferatum, F. solani and F. fujikuroi had faster mycelial development, with a growth rate of 7.72 - 8.27 mm/d, than F. thapsinum (6.52 mm/d). Conidia of F. oxysporum, F. proliferatum and F. solani were longer (4.74 - 5.96 µm) than those of F. fujikuroi and F. thapsinum (3.20 - 4.04 µm). Fusarium solani and F. oxysporum, respectively, had the largest and most partitioned conidia.


Introduction
Onion is produced all over the world. Its production in West African countries Despite the nutritional and economic importance of onion, its cultivation faces several biotic and/or abiotic constraints limiting its production. Fungal diseases are responsible for the estimated yield losses between 30% and 40% of the crop [5] and are the main biotic constraints to production. Diseases such as basal bulb rot are mainly caused by fungi of the genus Fusarium, also known to be responsible for seedling damping-off [6]. Fusarium oxysporum is the bestknown, pathogenic species responsible for wilting, root rot and crown rot on a variety of crops and often resulting in significant production losses [7]. Several authors have reported that F. oxysporium is the causal agent of basal onion rot [6] [8]. Zlata et al. [9] and Ghanbarzadeh et al. [10] have highlighted the responsibility of F. solani in the decay of the basal parts of onion plants leading to their sudden death. The importance of Fusarium species in onion cultivation in Burkina Faso has been poorly investigated. The only works are those of Dabiré [11] which have highlighted the involvement of F. oxysporium and F. solani in damping-off on seedlings and rotting of underground parts and bulbs in conservation. Accurate knowledge of the identity of a pathogen responsible for a given disease is the first step towards implementing adequate disease control and surveillance measures [12]. The identification of Fusarium species is made difficult because of the absence of discriminatory cultural or morphological characteristics. However, the molecular tool, particularly DNA sequencing, has emerged as a key means of identifying pathogenic fungi regardless of their stage of development and their morphology.
The objective of this study was to identify and characterize at the molecular and biometric levels the main species of the genus Fusarium associated with onion plants in field in Burkina Faso in order to determine those involved in the major diseases of the crop and develop an appropriate and sustainable management method for these pathogens.
Fusarium identified per plant, a small portion of mycelium was aseptically grown in a Petri dish containing PDA (Potato Dextrose Agar) culture medium. Successive transplantation of the isolates into the PDA medium has resulted in pure cultures of these fungi.

Molecular Identification of Fusarium Species Associated with Onion Plants in Burkina Faso
Monosporic cultures were produced from the pure cultures of Fusarium isolates.
For each isolate, two drops of conidial suspension containing approximately 100 spores/ml of suspension prepared from a 10 day-old pure culture were spread on an Agar medium contained in a Petri dish. Two days after seeding, the germinating spores were transferred to Petri dishes containing the PDA medium using only one germinating spore per dish, and left to grow. Three monosporic isolates were produced per isolate. Based on the color of the mycelium and/or the shape of the conidia, one to four monosporic isolates were selected per site for molecular analysis. The DNA was then determined by spectrophotometer (Nanodrop 2000) and the initial concentration evaluated in nanograms per microliter (ng/µl). The different concentrations were diluted on a case-by-case basis so that the volume of DNA (3 µl) collected for PCR contains about 100 ng of DNA. The amplification reaction was performed in 25 µl containing 100 ng DNA (3 µl); 2.5 µl BD buffer, 10×; 0.5 µl dNTP (10 mM); 2 µl MgCl (25 mM); 0.2 µl Taq polymerase; 15.8 µl sterile distilled H 2 O and 0.5 µl EF1 to 10 µM and EF2 to 10 µM primers. The primers used were: EF1: ATGGGTAAGGARGARGACAAGAC [15]; EF2: GGARGTACCAGTSATCATCATGTT [16].
The amplification reaction was performed according to the following program: a denaturation cycle at −95˚C for 10 minutes followed by 35 consecutive cycles (denaturation at 94˚C for 30 seconds, hybridization at 52˚C for 30 seconds and elongation at 72˚C for 45 seconds). The electrophoresis was performed on a 1% agarose gel at 120 V for 20 minutes. The amplification products were visualized under UV light, using ethidium bromide previously incorporated into the agarose gel. Thirty microlitres (30 µl) of PCR product from each isolate were collected for sequencing.
The sequencing was carried out by Genewiz [17], with the P363 and CHOO9 primers. For the identification of Fusarium species, the sequences were processed, cleaned and aligned with the Chromas software. The consensus sequences obtained were compared with those in the NCBI (National Center for Biotechnologies Information) database on the site [18].

Biometric Characterization of Fusarium Species Associated with Onion Plants in Burkina Faso
In order to determine morphological and cultural characteristics for a good differentiation of Fusarium species isolated from onion plants, three biometric characteristics related to the colony growth in culture, the conidia sizes and the number of cells (or divisions) per conidia were evaluated. The experimental design used is a randomized complete block design with four replications. To assess the mycelial growth of the colonies, the diameter of the colonies was measured in each dish on the ninth (9th) day after the incubation of the explants in the PDA medium. The result, expressed in millimeters, was the average of two diameters perpendicular to each other.
The mycelial growth rate was calculated according to the formula: The biometric data collected were subjected to an analysis of variance using the Statistical Analysis System (SAS) software, version 8; 2001. The calculation and separation of the means of the measured parameters were performed according to the Duncan test (Duncan Range Multiple Test), at the 1% threshold.

Prevalence of Fungi of the Genus Fusarium on Onion Plants in Different Growing Sites in Burkina Faso
A total of 510 onion plant samples were collected from the 17 production sites.
Visual examination of these incubated plant samples allowed the detection and isolation of several types of Fusarium associated with the plants. The results of the analysis of variance showed that the prevalence of fungi of the genus Fusarium varied widely (p < 0.0001) depending on the collection sites ( Table 1). The fungus was present in all onion growing sites where it infected 10% to 90% of the sampled plants with an average attack rate of 62.55%. The sites studied were classified into four distinct groups: Group 1, composed of nine (9) sites (Yako, . Considering the onion production areas, the results also indicated that the prevalence of Fusarium was significantly higher (p = 0.0004) in the northern, western and central zones of the country than in the southwestern zone ( Table 2).

The Main Fusarium Species Associated with Onion Plants in Burkina Faso
Based on the color of the mycelium, the shape and/or the size of the conidia, thirty-six monosporic isolates were selected for molecular analysis. Chromatography results obtained on agarose gel using the primers EF1 and EF2 indicated that the molecular weights of the different products are approximately 700 bp for all isolates (Figure 2), indicating that these isolates are indeed fungi belonging to the genus Fusarium.
Analysis of the obtained sequences and their comparison with the sequences in the NCBI database revealed 99% of identity for 26 isolates, 98% for five isolates, 97% for four isolates and 93% for one isolate (Table 3). The analysis showed that the isolates belong to five species of Fusarium: -Fusarium oxysporum which includes 16 isolates (44.44% of the studied isolates) with similarity rates of 93% -99% with the reference accessions JF957830.1, KU985430.1 and JF957820.1 of the NCBI.
-Fusarium fujikuroi which also includes two isolates (5.55%) with similarity rates of 97% to the NCBI reference accession LC055826.1.

Biometric Characteristics of the Main Fusarium Species Identified on Onion Plants in Burkina Faso
The analysis of variance performed on biometric data including the mycelial growth speed of fungi, the length and diameter of conidia, the number of cells per conidium, revealed significant differences between the Fusarium species. In general, F. oxysporum, F. proliferatum, F. solani and F. fujikuroi had rapid mycelial development (7.72 -8.27 mm/d) on the PDA culture medium compared to F. thapsinum which had slow growth (6.52 mm/d) (  In the F. proliferatum species, 12 isolates (i.e. 80%) grew rapidly (7.86 -9 mm/d) against three isolates (i.e. 20%) with slow growth (Table 6). Among the fast-growing isolates, F.pro-4-Sm; F.pro-15-R; F.pro-3-Sm produced long conidia (9.44 -11.24 µm) with 2.60 to 2.96 cells each (Table 6). In contrast, the other isolates had small conidia.
F. fujikuroi included two isolates, one fast-growing (8.98 mm/d) and the other slow-growing (6.81 mm/d). For the other characteristics, no significant differences were noted between these two isolates ( Table 7). In F. solani, the isolate F. s-38-Tg was characterized by a rapid growth rate (9 mm/d) and large conidia (2 µm wide) and the isolate F. s-24-Kk by a slow growth of 6.45 mm/d and fine conidia (1.76 µm wide).

Discussion
The results of the analysis of the onion plants sampled in seventeen onion growing sites in Burkina Faso has allowed to note the presence of fungi of the genus Fusarium in these sites and to classify these sites into three categories according to the proportions of plants contaminated by these fungi. Fusarium was found in all the studied sites, indicating that the fungus is present in onion fields , each with three to six cells. The conidia of these two isolates appear to be predominantly macro conidia unlike the other isolates of the species whose conidia are small in size, therefore micro conidia. Similarly, for the isolates F.pro-4-Sm; F.pro-15-R; F.pro-3-Sm of the species F. proliferatum which produced long conidia (9.44 -11.24 µm) having 2 to 3 cells each, their conidia appear to consist of macro conidia. For the other isolates, the majority of the conidia produced which are small in size (<10 µm long) should be mainly formed by micro conidia. These results suggest the existence of different strains within the species F. oxysporum and F. proliferatium. The present study confirms that of Sumana et al. [22] who detected a strong genetic diversity in F. oxysporum f.

Conclusion
Fungi from the genus Fusarium have been detected on onion plants in Burkina Faso at rates ranging from 10% to 90%. Molecular analysis of 36 Fusarium isolates collected from different onion production sites identified five Fusarium species including F. oxysporum, F. proliferatum, F. solani, F. fujikuroi and F. thapsinum. Fusarium oxysporum and F. proliferatum, which accounted for 44.44% and 41.46% of isolates, respectively, were the most representative species K. R. Kintega et al. in all the production areas. The bio-morphological study showed that F. oxysporum, F. proliferatum, F. solani, F. fujikuroi had faster mycelial growth on PDA medium than F. thapsinum. The identified Fusarium species, in particular, F. oxysporum and F. proliferatum, regrouped within them, isolates whose conidia were predominantly composed of macro conidia and other isolates whose conidia were mainly composed of micro conidia. These results suggest the existence of different strains within each of the Fusarium species. A pathogenic characterization of the different isolates would allow, on the one hand, to identify the Fusarium species involved in seedling damping-off and basal bulb rot, two major pathologies of onion in Burkina Faso, and on the other hand, to determine the most pathogenic Fusarium strains. Precise knowledge of these pathogens is essential for the development of adequate and sustainable management methods for these diseases.