CHARACTERIZATION OF FUSARIUM SPECIES ASSOCIATED WITH MANGO MALFORMATION DISEASE IN SOUTHERN SENEGAL

The first clade is made up of 4 sub-clades where all the isolates studied are present except 12-Ziguinchor, 2 and 11 Kolda isolates. The firstsub-clade with a bootstrap value less than 50%, formed by the isolates contig 5 Kolda, contig 8 Kolda and contig 7 Kolda are closer to KF576628 1 Fusarium sterilihyphosum EFA 6FSRG isolate from the NCBI database as well as the contig 53 Sedhiou, 3 Ziguinchor, 26 Sedhiou, 6 Kolda and contig 61 isolates which do not belong to any subclade. The second subclade with a bootstrap value of less than 50% formed by contig 9 Kolda and the reference strain of the NCBI Fusarium sterilihyphosum database. The third subclade consisting of MK226291. fujikuroi Fusarium fujikuroi verticillioides And fourth subclade bootstrap value 99% reference NCBI equiseti

In Senegal mango production has long existed in traditional forms, especially in the south and center of the country. It accounts for 60% of the country's fruit production, with an estimated annual output of 150,000 tons. Despite a positive balance sheet in recent years, mango orchardsareaffected by numerous phytosanitary constraints like diseases and insect pests. Among the diseases, mango malformation is considered one of the most deadly for the mango tree. Since its discovery in 2009 in the south of Senegal, the disease has continued to spread in this area with incursions towards the northern part of the country. Few studies were dedicated to that pathogen in the Senegalese context. This study was carried out to help bring more light in the identity of the pathogen(s) causing this disease. Therefore 13 isolates of Fusarium isolates obtainedfrom mango malformation tissueswere characterized using morphological criteria and the molecular analysis. The 5.8S rDNA region of the internal transcribed spacer (ITS) was amplifiedusing ITS1 and ITS4 primer pairs. Sequence analysis and other analytical studiesshowed that the malformation of mango tree in southern Senegal is relatedto a diversity of Fusarium species. The morphological and molecular analysisallowed to identify several Fusarium strains with a very high degree of similarity (99.08 to 99.82%) with the species in the NCBI database ranged.  323 Mangoes are grown in most West African states, with a production area of 540,000 km², stretching from Senegal through western Gambia to eastern Nigeria (Ministère du commerce, 2016). Several countries in the subregion, including Senegal, are currently using it as their main export activity (CARE, 2009).
In Senegal mango production has long existed in traditional form, especially in the south and center of the country. The reduction of the main cash crops, such as groundnuts and millet, has led farmers to seek alternative incomegenerating activities. To this end, some producers in the south and center have seen the modernization of traditional orchards and the creation of new mango plantations for export. Since then, mango production accounts for 60% of the country's fruit production, generating an estimated annual output of 150,000 tons (Diedhiou et al., 2014). The mango sector becomesthe most dynamic in fruit exports in the country (Diouf, 2016).Mango exports to the European Union and the countries of the subregion increased from 300 tons in 1998 (Rey, 2011) to 16770 tons in 2015 (Diouf, 2016).
The mango-producing regions are : Dakar, Thies, Saint-Louis, Fatick, Kolda, Ziguinchor and Sedhiou (Diedhiou et al., 2014). However, despite a fairly positive balance sheet in recent years, mango production is affected by numerous phytosanitary constraints, including mango malformation disease. It is probably considered as the most crucial disease of mango at present (Nafees et al., 2010;Kumar et al., 2011),not only because of its widespread and destructive nature, but also because of its confused etiology since its discovery despite several years of research. Economic losses have been estimated in several countries to be between 10-90% (Kumar et al., 2016). The mango malformation disease manifests itself as a vegetative malformation and a floral malformation that directly reduces yield because the affected panicles become sterile (Kumar et al., 2011;Freeman et al., 2014a). Several abiotic and biotic stresses are associated with mango malformation disease. Following several years of continuous research, fungi of the genus Fusarium spp. have been most often reported as the main cause of this disease (Iqbal et al., 2010;Iqbal et al., 2011;Freeman et al., 2014a;Kumar et al., 2016). Several species of Fusarium have been identified worldwide. The disease was first recorded in West Africa in southern Senegal in 2009 in Tandouk in the department of Bignona (Senghor et al., 2012). Subsequently it was also reported in Mboro in Thies region in 2012 (Senghor et al., 2012). The disease has been progressing steadily in the South since its discovery where the incidence of the disease ranged from 20 to 91% (Dieye et al., 2020). Thus, it is urgent to know all the Fusarium species associated with mango malformation disease in southern Senegal. The morphological characterization of Fusarium species is very complex despite their differences at the colony level, the forms of macroconidia, the presence or absence of microconidia, chlamydospores and the formation of secondary structures are very delicate criteria to differentiate in some cases (Leslie et Summerell, 2006). This study aims to identify the Fusarium species responsible for mango malformation usingmorphological characterization as well as molecular analysis of 13 Fusarium isolates obtained from samples of malformed mango sampled fromsouthern regions of Senegal.

Study site:
Samples were taken from the roads in the southern regions (Tambacounda, Kolda, Sedhiou and Ziguinchor) of Senegal ( Figure 1). The climate is of the Guinean Soudano type and characterized by heavy rainfall during the rainy season.

Sampling:
Sampling was carried out every 50 ± 5 km on 30 mango trees at a rate of 10 feet chosen at random in 3 directions per locality from Velingara to Ziguinchor and Ziguinchor to Diouloulou (Figure 1).

Isolation and purification:
The samples(floral and vegetative malformations) were first disinfected with 1%. Then, using a sterile scalpel blade, explants were taken. Under the fume hood, these explants were successively immersed in 70% alcohol, rinsed with sterile distilled water, wiped with sterile absorbent paper and finally inoculated with 3 explants per Petri dish containing the PDA medium. The plates are incubated in the oven at 25°C.Few days after, some of the mycelium is transferred to other plates to obtain pure strains.
Single-spore culture protocol: It was carried out using Booth's 1971 protocol to obtain genetically homogeneous fungal material (Booth, 1971). For this purpose, the isolates were first transplanted to Petri dishes containing PDA medium and allowed to grow over the entire surface of the dish for 7-10 days. A 5mm diameter mycelial disc was taken from the periphery and introduced into a tube containing 9ml sterile distilled water. After shaking, dilutions to one hundredth are made with a sterile pipette. This operation is repeated as many times as necessary until the desired dilution is reached. Then a volume of 0.1ml generally containing 2 to 5 microconidia adjusted with the Thomas cell was taken from the last 2 dilutions and spread with sterile beads on a 2% agar medium. Identification and delimitation of germinating spores was performed using a binocular magnifying glass after 48h incubation at 25°C. Fragments of 3 to 4 of these conidia are collected with their agar using a sterile loop and placed in Petri dishes containing PDA before being incubated at 25°C for one week. This operation was repeated for all the different isolates. This operation was repeated for all the different isolates.

Study of morphological variability:
To study the morphological variation between different Fusarium spp. isolates, each isolate is transferred to a Petri dish containing a PDA medium combined with chloramphenicol to avoid bacterial contamination. The inoculation was done using a 5mm diameter mycelial disc taken aseptically with a fume hood punch from a 10-day old Fusarium sp. culture before incubation at 25°C. The daily evolution of the mycelial diameter of the different isolates was regularly done for each replicate. The mycelial diameter was measured on the horizontal and vertical planes of the box and the average of the two measurements was calculated. This procedure was carried out until the mycelium had grown over the entire surface of the box.

Morphological characterization:
The macroscopic study was done based on the colour on the surface and reverse side of the boxes, the growth rate of the mycelium and the pigmentation. While the microscopic description was done by observing the shape of the microconidia, macroconidia, phialides and chlamydospores. In addition, a biometric study was carried out on 50 conidia per isolate.

Molecular characterization:
Molecular identification was done using the regions of the internal transcribed spacer. DNA was extracted from mycelia grown on Petri dishes containing potato dextrose agar (PDA) medium. DNA extraction of each isolate was performed using the BIOLINE Isolate II Plant DNA kit according to the kit protocol. Then aNanodrop assay was performed prior to PCR. A PCR reaction was prepared for all 13 DNA samples according to the following protocol using ITS1 / ITS4 primers.
PCR products were detected by migration electrophoresis on 1% agarose gel in the presence of a 1Kb molecular weight marker. The photos were visualized by the photo documentation system "G Box". The PCR products from the amplified DNA samples were sent to the sequencing laboratory for further analysis.
After sequencing and processing by BLAST, the consensus sequences were compared to sequences in the NCBI GenBank database. The identificationof each isolate was defined based on the closest percentage resemblance to the Blast results. A phylogenetic tree was then constructed using MEGA 10.1.6 software (Kumar et al., 2018) using the Maximum Likelihood algorithmwith 1000 bootstrap réplications (Felsenstein, 1985). The resulting sequences were subsequently submitted to the NCBI for accession number assignment.

Discussion:-
Morphological characterization of the strains isolated from the mango floral malformation in southern Senegal has made it possible to observe different fungal structures. These are microconidia, chains of microconidia, hyphae, macroconidia, monophialides, polyphialides and chlamydospores, typical of Fusarium (Leslie et Summerell, 2006) including some species associated with mango malformation disease identified by (Britz et al., 2002;Senghor et al., 2012). These results confirm that mango malformation disease in southern Senegal is associated with a species complex of the genus Fusarium. With the exception of F.equiseti, which belongs to the Gibbosum section, and F.oxysporum, all other species belong to the Gibberella section. Thus, the isolates showed cultural and microscopic similarities with the species Fusarium mangiferae, Fusarium subglutinans, Fusarium oxysporum, Fusarium 329 sterilihyphosum, Fusarium equiseti and an isolate which could not be assimilated to any of these species: mycelia sterilia.
It should be noted that the morphological characterization of Fusarium spp. is very complex. The culture criteria used allowed aprovisional identification of the species. However, this identification may be influenced by a number of parameters, namely the composition of the culture medium, the purity of the culture of the isolates studied, the incubation conditions, etc. According to Leslie and Summerell, (2006), some species producing chlamydospores may be very close to those that do not produce chlamydospores as these spores are not conserved over time. In 2012, species isolated from mango malformation in southern Senegal were very close to F. mangiferae, F. sterilihyphosum and F. tupiense following morphological characterization. In this study, isolates 7 and 26 were also compared to F.sterilihyphosum.
With probably due to the fact that the genus Fusarium is a genus that is evolutionarily recent and therefore unstable, hence the importance of the number of species and the variations between species on the same plant and from one plant to another in this genus.
The abundance of one species relative to another can also variesfrom one region to another depending on environmental conditions, including anthropogenic activity, presence or absence of sexual forms, stage of infection, and host plant receptivity conditions (Leslie et Summerell, 2011).
With regard to the results of the morphological characterization and molecular study, it should be noted that, despite the high degree of similarity between the Fusarium spp. isolates studied and the reference species in the NCBI database, morphological differences were also noted. In addition, most of the species selected as a result of morphological characterization did not coincide with those obtained during sequence comparison in the NCBI database, which shows the limitations of the methods used when further comparison is carried out. Even for molecular analyses there are limitations and there is also the problem of the repeatability of the results, especially since the method is essentially based on the search for similarity with already identified species.
It was interesting to see the relationships between the Fusarium spp. isolates studied and some reference species in the NCBI database in so far as mango malformation disease is associated with several species of the Gibberella fujikuroi complex such as F. subglutinans (O'Donnell et al., 1998); F. mangiferae (Britz et al., 2002), F sterilihyphosum (Britz et al., 2002), F. tupiense (Lima et al., 2012;Crespo et al., 2016) among others. Thus, the resulting phylogenetic tree revealed that isolates could show a high degree of similarity with a reference species in the NCBI database and be distant on the tree. This was found for most of the strains studied with the exception of isolate 17.
In the end, even with the results of the molecular characterization, it did not seem possible to identify exactly which Fusarium species corresponded to the isolates studied, although it did allow for greater accuracy. This could be explained by the fact that the primers used were not very specific and do not always allow to distinguish very close species due to the variability of ITS zones. But also that these species are very phylogenetically close. Thus, it would be important to be more precise in the molecular approach by using specific primers based on genes well represented in the database such as the β-tubulin and elongation factor -1α.

Conclusion:-
Thus, from this study on the characterization of isolates associated with mango malformation disease in Senegal, it appears that the malformation of mango in Senegal is due to species of the genus Fusarium which are phylogenetically very similar. One morphologically closer to Fusarium mangiferae is probably the most implicated in the disease while the others are thought to play a complementary role in the infection process of mangoes, as is the case for other plant crops attacked by Fusarium.