1 Influence of soil mycoparasites on tomato cultivation (Solanum lycopersicum) in the locality of

2 Songon in Côte d’Ivoire.

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3 Sagou Dominique a *, Pakora Gilles Alex a , Amari Ler-N'Ogn Dade George Elisée b

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4 a Laboratory of Biology and Health, UFR of Biosciences, Félix Houphouët-Boigny University, 22

5 BP 582 Abidjan 22, Ivory Coast,

6 b Biotechnology Laboratory, Agriculture and development of biological resources, UFR of

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7 Biosciences, Félix Houphouët-Boigny University, 22 BP 582 Abidjan 22, Ivory Coast,

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8 *Author corresponding to: Laboratory of Biology and Health, UFR of Biosciences, Félix

9 Houphouët-Boigny University, 22 BP 582 Abidjan 22, Ivory Coast

10 (+225) 0747969858 [email protected]

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21 Abstract

22 Faced with the high demand for market garden crops, urban and peri-urban farmers use several

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23 cultural practices to control phytopathogens, including bioproducts. The objective of this study was

24 to study the influence of microscopic fungi isolated from tomatoes from two different habitats. Thus,

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25 the material used in this work consisted of tomato leaves from two different habitats. The

26 methodology used during this study consisted of isolating endophytic fungi from the leaves of this

27 plant on Potato Dextrose Agar medium. The isolates obtained were subjected to macroscopic,

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28 microscopic and molecular identification by amplification of the ITS regions of their ribosomal DNA.

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29 A total of 17 endophytic fungi were isolated from both sides of the tomato plants and characterized

30 according to the genus Aletrnaria, penicillium, Nigrospora, Curvularia, Cladosporium,

31
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Pestalotiopsis, Trichoderma . Microbiological and molecular analyses showed that the genera

32 Nigrospora and Curvularia are the most isolated. The species Alternaria burnsii and
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33 Neopestalotiopsis rosea isolated from tomato leaves grown in untreated soil are recognized as latent

34 pathogens and are responsible for biotic stress in plants in general and tomatoes in particular.

35 Keywords: Solanum lycopersicum , endophytic fungi, soil, Songon- agban , Ivory Coast
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44 1. Introduction

45 tomato plant (Solanum lycopersicum L.,1753) is a species of plant herbaceous plant of the Solanaceae

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46 family , sensitive to cold, perennial in warm climates, generally annual, native to the northwest of

47 America of South . Intended for fresh consumption or industrial processing . According to FAO

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48 statistical data (FAO, 2018), global tomato production broke records during the year 2018; At the top

49 of the ranking, China with a volume of tomato produced of 60,540,758 tons, or 31.8 % of world

50 production. As for Ivory Coast, it occupies 103rd place . world ranking with an estimated production

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51 of 40,560 tonnes which represents a negligible global share. In Ivory Coast, the tomato is the most

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52 cultivated vegetable in rural, urban and peri-urban areas. Its cultivation constitutes an income-

53 generating activity for the populations, particularly women (Adje et al., 2010) However, the

54
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numerous parasitic attacks of certain microorganisms make it difficult to grow tomatoes in tropical

55 countries in general and in Côte d'Ivoire in particular (Soro et al., 2008). Among these parasitic
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56 diseases, white collar rot caused by Sclerotium rolfsii is the most harmful, leading to a significant

57 drop in tomato production and a deterioration in its quality (Soro et al., 2008). Necrotrophic species

58 of the genus Alternaria responsible for the disease alternaria can cause yield losses of around 90%
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59 due to its mycotoxins such as alternariol (Sikirou et al., 2007; Adje et al., 2010; Adhikari et al.,

60 2017). This is also the case for the genus Colletotrichum associated with tomato anthracnose
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61 worldwide. Furthermore, The control of these phytopathogens involves several methods of control,

62 including chemical and biological. Thus, to combat plant diseases in general and tomatoes in
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63 particular, farmers commonly use synthetic fungicides (Pohé , 2012) . Because these have the effect

64 of considerably reducing these pathogens. In addition to these control methods, cultivation techniques
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65 applied to the soil could be useful in order to improve not only soil quality but also the growth of

66 tomato crops. In this context, the general objective of this study is to study the influence of

67 mycoparasites isolated from tomatoes from two different habitats.

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69 2. Material and method

70 2.1 General

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71 TLC was performed on pre-coated silica gel plates (Silica 60 F254 Merck, Germany). 1D and 2D

72 NMR spectra were recorded in DMSO-d6 with Bruker 400 spectrometers using TMS as an internal

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73 standard. Chemical shifts were given in δ (ppm). HR-ESI-MS and fragmentation MS spectra were

74 obtained with an Applied Biosystem QSTAR Pulsar I mass spectrometer.

75

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76 2.2 Plant material

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77 The plant material consists of tomato leaves taken from plants grown in the greenhouse of the

78 Industrial Research Unit (URI) "Biopesticides" section located at the Scientific and Technological

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Innovation Center of Bingerville, Côte d’Ivoire. Tomato crops were grown on soil from the

80 production area with high pest pressure located in Songon-Agban in the south of Côte d’Ivoire until
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81 the vegetative stage. Among the pathogens, Sclerotium rolfsii is the most common, which causes a

82 lot of damage to tomato crops in Côte d’Ivoire.

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83 2.3 Methods
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84 2.4 Soil treatment

85 A portion of the soil collected from the Songon-Agban tomato production site was autoclaved at 121
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86 °C for 15 min. Thus, two batches of tomato crops were carried out on two types of soil. The first

87 batch of seeds was placed on treated soil and the second batch was placed on untreated soil. The

88 tomato crops carried out were monitored for up to 30 days at the vegetative stage.
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89 2.5 Processing of harvested leaves and isolation endophytic fungi

90 Apparently healthy and mature leaves were randomly collected one leaf per plant from different
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91 locations. They were placed in sterile plastic bags and transported to the laboratory for further use.

92 The samples thus collected were stored at a temperature of 4 °C until use. The isolation of endophytic
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93 fungi was done according to the following protocol: Tomato leaves were rinsed with tap water for

94 about ten minutes to remove impurities and surface debris . Then, they were immersed in 70% ethanol

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95 for 1 minute to fix epiphytic microorganisms on the leaf surface, then in sodium hypochlorite

96 (NaOCl) (3%) for 4 minutes; the leaves were then put back in 70% ethanol for 30 seconds, and were

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97 rinsed three times with sterile distilled water for one minute each time and dried on sterile filter paper.

98 The treated leaves are cut into fragments of a few millimeters (5 mm) at a rate of 3 to 5 segments per

99 dish and placed aseptically in Petri dishes containing Potato Dextrose Agar (PDA) previously

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100 autoclaved at 121 ° C for 15 minutes, and supplemented with 150 mg / L of chloramphenicol to inhibit

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101 bacterial growth. The dishes are incubated at 27 ° C for three days. ( Hazalin et al., 2009 ; Khan et

102 al ., 2010)

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2.6 Identification of isolated endophytic fungi

104 2.6.1 Macroscopic and microscopic identification

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105 Pitt's identification keys and Hocking, 1985; Botton et al., 1990; Kidd et al. 2016 were used. On a

106 slide containing a drop of water, a mycelial explant was spread and then observed under a microscope

107 at x10, Gx40 and Gx100 magnification. As for the macroscopic characteristics, they concerned the
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108 color and shape of the colonies and the speed of propagation of the mycelium.
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109 2.6.2 Molecular identification

110 2.6.2.1 Extraction of genomic DNA from isolates

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111 Genomic DNA extraction from microscopic fungi was performed according to the method described

112 by (White et al., 1990) with some modifications. The liquid culture of the fungi previously made
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113 with PDB medium was centrifuged at 10,000 g for three (3) min. After removal of the supernatant,

114 the pellet was homogenized in 400 µL of TBS buffer (0.4 M NaCl, 10 mM Tris-HCl at pH 8 and 2

115 mM EDTA at pH 8) and then vortexed for one minute. Then, 40 µL of 20% SDS and 8 µL of
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116 Proteinase K (20 mg/mL) were added, and the mixture was incubated at 60°C for 3 hours. After

117 incubation, 300 µL of 6 M NaCl were introduced into the different tubes, which were also vortexed
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118 for 30 seconds and then centrifuged at 10,000 g for 30 min at 4°C. The supernatants were transferred

119 to 2 mL tubes to which an equal volume of 100% Isopropanol was added. These tubes were incubated

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120 at -20°C for one hour and then centrifuged at 10,000 g for 20 min at 4°C. The pellet was washed with

121 70% ethanol and then centrifuged again for 10 min at maximum speed. The pellet from the

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122 centrifugation was subsequently recovered and dried at 37°C for 5 min. The DNA thus obtained was

123 resuspended in 300 µL of sterile TE buffer (Tris-EDTA) and then stored at -20°C for amplifying.

124 2.6.2.2 Electrophoresis of DNA bands

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125 Electrophoresis was performed with a 1% agarose gel prepared with TBE (Tris, Borate, EDTA) buffer

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126 at 1X. The gel was cast (10 mL) on a plate with an eight (08) well comb. After solidification, it was

127 introduced into the SYBR SAFE (1%) for 30 min, before being placed in the migration tank

128
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containing TBE migration buffer (1 X). A volume of 3 µL of crude DNA and 3 µL of loading buffer

129 were subsequently deposited in the wells provided for this purpose. The migration was carried out in
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130 a Mupid at 100 volts for 10 min. The quantity of DNA is assessed by comparing the brightness of the

131 band to that of the molecular weight standard.

132 2.6.2.3 Amplification by Polymerase Chain Reaction (PCR)

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133 It is an enzymatic reaction that allows the chain amplification of a specific region of DNA. Its
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134 principle is based on the in vitro enzymatic amplification of a segment of DNA using oligonucleotide

135 primers complementary to the sequences of the segment to be amplified in 5' and 3'. The molecular

136 identification of fungi was carried out based on the sequences of ribosomal DNA. Indeed, the
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137 ribosomal unit present in the genome of all organisms is repeated in tandem in fungi. The sequences

138 of this unit contain both variable regions and conserved regions . Thus, the ITS (Internal Transcribed
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139 Spacer) region of ribosomal DNA is a conserved area within the same fungal species, but which varies

140 from one species to another. PCRs were performed in a 23.5 µL reaction volume containing 5 µL of
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141 DNA template, 4 µL of Premix (5x Firepol Master MIX), 13 µL of bio-mol water and 1.5 µL of

142 primer pairs ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3'), ITS 4 (5'TCCTCCGCTTA

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143 TTGATATGC-3') ITS 5 (5'-TCCGTAGGTGAACCTGCGG-3') (White et al., 1990; Gardes and

144 Bruns, 1993). Negative controls (without DNA) and positive controls (with DNA) were performed.

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145 Amplification of ITS regions of fungal DNAs was performed according to the following program: an

146 initial denaturation of 94°C for 4 min, followed by primer annealing time of 94°C for 1 min, 55°C

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147 for 1 min and 72°C for 2 min for 35 cycles. A final elongation of 72°C for 15 min.

148 2.7 Direct confrontation test on agar

149 In order to select among the fungi isolated from the untreated soil, those that have the capacity to

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150 inhibit the mycelial growth of Sclerotium rolfsii in order to use them as a biocontrol agent. Thus, on

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151 a PDA agar a mycelial explant of approximately 6 mm in diameter of each fungus against that of

152 Sclerotium rolfsii were confronted for 10 days. After this time interval, an observation was made to

153
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assess the quality by measuring the inhibition diameters and the antagonistic power of the fungi. For

154 this, the formula proposed by Houmni et al., 1996 was used.
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I (%) = (1-Cn/Co) x100

156 C n = average diameter of colonies in the presence of the antagonist

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157 C o = mean diameter of control colonies (pathogen)

158
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159 2.8 Purification of secondary metabolites from Alternaria burnsii and Neopestalotiopsis rosea

160 In order to know the mycotoxins produced by these fungi, a chemical study was carried out on the
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161 two mycoparasites recognized as phytopathogens of plants. A volume of 2 mL of a spore suspension

162 prepared in sterile distilled water of Alternaria burnsii and Neopestalotiopsis rosea was distributed
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163 on sterilized rice medium (40 g of rice and 40 ml of distilled water) and incubated at 28 °C for 21

164 days. At the end of the 21 days of incubation, the cultures of these fungi were macerated and extracted
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165 with ethyl acetate. The organic phases were combined, dried (MgSO4) and evaporated under vacuum

166 at 35 °C. The crude extract obtained was subjected to silica gel column chromatography using an

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167 elution composed of a system Cyclohexane 100: Ethyl acetate 0 to Cyclohexane 20: Ethyl acetate 80.

168 The isolated secondary metabolites were subjected to LC/MS and NMR analysis to determine their

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169 molecular mass and chemical structure, respectively.

170 3. Results

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171 plant leaves

172 Tomato leaves collected from tomato plants grown on heat-treated soil (Fig. 1a) were greenish in

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173 color and appeared more vigorous with very good vegetative growth of tomato plants. On the other

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174 hand, tomato leaves on untreated soil (Fig. 1b) had a brown appearance and delayed vegetative

175 growth of the plants. It appears that tomato plants obtained on heat-treated soil had very good

176 vegetative growth with better chlorophyll pigment unlike tomato plants grown on untreated soil. The

177
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latter showed a delay in development with a yellowish-red pigment at the level of the leaves.
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178
A B

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183 Fig. 1: Appearance of tomato leaves: a - on treated soil; b - on untreated soil

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185 3.2 Identification of endophytic fungi isolated from tomato leaves

186 After 3 days of incubation, mycelial growth was observed from sections made on leaf segments
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187 cultured on the third day. The thin filaments were placed on PDA agar medium. The agar Petri dish

188 was invaded by endophytic fungi. The isolates were purified on new PDA agar Petri dishes until pure

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189 cultures were obtained. After purification of the isolates, a diversity of endophytic fungi was isolated

190 from both sides of Solanum lycopersicum grown on treated and untreated soil. The fungal isolates

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191 could be identified initially macroscopically and microscopically and then biologically. molecular .

192 Macroscopic and microscopic identification

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193 A diversity of fungi was isolated and identified according to the identification keys taken from the

194 literature. Thus, several genera were counted, namely:

195 the genus Nigrospora

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196 A total of 6 fungi of the genus Nigrospora were counted and described as follows: The colony on agar

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197 gives a whitish woolly appearance and grows quite rapidly . The conidia observed are black and shiny

198 which stand out in sharp contrast giving the colonies a striking appearance under the binocular
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199 dissecting microscope. In older cultures the hyphae darken and the colonies appear black, with

200 abundant conidia production. The conidiophores are short, pale brown, swollen and borne
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201 perpendicular to the hyphae, bearing single and terminal conidia. The conidia are smoky brown or jet

202 black, spherical or ovoid. Fig. 2 (isolates 2;3;6 and 9); Fig. 3 (isolates 2 and 6).
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203 The genus Curvularia

204 A number of 6 fungi of the genus Curvularia have been isolated and described as follows. The
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205 colonies are fast growing, downy type, brown to blackish brown. The conidiophores are erect,

206 straight, septate, often geniculate sometimes nodular. The conidia are ellipsoidal, often curved or
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207 lunate, rounded at the ends or sometimes slightly tapered towards the base, pale brown, reddish brown

208 medium to brown dark Fig. 2 (isolates 5 and 7); Fig. 3 (isolates 1;3;4 and 5).
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209 The genus Penicillium

210 A fungus of the genus Penicillium has been isolated and described as follows: The colony is usually

211 fast growing, green in colour, sometimes with a white margin. Microscopically, chains of single-
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212 celled conidia are produced in basipetal succession from a specialised conidiogenous cell called a

213 phialide. These chains of conidia where the youngest conidium is at the basal or proximal end of the
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214 chain. In Penicillium, phialides may be produced singly, in groups or from branched metulae, giving

215 a brush-like appearance (a penicillus) Fig. 2 (isolate 4).

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216 The genus Alternaria

217 A fungus of the genus Alternaria has been isolated and described. The colony is fast-growing, olive

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218 or grayish in color, and suede-like or flaky. Microscopically, branched acropetal chains

219 (blastocatenary) of multicellular conidia (dictyoconidia) are produced sympodially from simple,

220 sometimes branched, short or elongated conidiophores. The conidia are obclavate, obpyriform,

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221 sometimes ovoid or ellipsoidal, often with a short conical or cylindrical beak, pale brown, smooth or

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222 warty-walled. Fig. 2 (isolate 11).

223 The genus Pestalotiopsis er

224 A fungus of the genus Pestalotiopsis: the colony is whitish in color. The conidiophores (annellides)

225 are generated in compact fruiting structures (aecervuli or pycnidia). The spores (conidia) are 4- to 5-
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226 celled, with the two or three central cells dark brown, and with two or more hair-like apical

227 appendages; they cluster in a moist mass Fig. 2 (isolate 8).

228 The genus Trichoderma

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229 A fungus of the genus Trichoderma: The genus is usually recognized by its rapid growth producing
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230 white, green, yellow colonies of cushions of sporulating filaments. The fertile filaments or

231 conidiophores produce whorled lateral branches bearing short phialides. The single-celled spores

232 (conidia) are produced successively, starting from the tips of the phialides and grouped in small moist
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233 masses Fig. 2 (isolate 10).

234
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235 The genus Cladosporium

236 A fungus of the genus Cladosporium: Conidiophores are macronematous, straight or slightly
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237 flexuous, distinctly nodose, pale or semi-pale brown, smooth. Conidia arise from terminal swellings,

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238 which later become intercalary, in simple or branched chains. Conidia are cylindrical, rounded at the

239 ends, ellipsoidal, limoniform or subspherical, subhyaline. Fig. 2 (isolate 1).

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 Isolate 4 Isolate 1 Isolate 2

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Isolate 9

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Isolate 7 Isolate 8

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Isolate 3

r Isolate 6

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Isolate 5

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Isolate 10
o Isolate 11

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 Isolate 5 Isolate 3 Isolate 4 Isolate 6

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Isolate 2 Isolate 1

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243
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244 Fig. 3 : Endophytic fungi isolated from tomato leaves grown on treated soil

245
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246 Molecular identification

247 The isolation of endophytic fungi led to a diversity of fungal isolates. After amplification of DNA by
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248 PCR based on the ITS regions of their risomal DNA. A total of 17 genera of endophytic fungi were

249 counted including 11 isolates on untreated soil (Fig. 2) and 6 isolates on treated soil (Fig. 3) . Thus,

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250 the size of the DNA bands obtained after gel migration were determined according to the molecular

251 weight. The BLAST of the sequences obtained in the NCBI GENEBANK allowed to identify the

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252 fungal isolates from the percentage of identity and under their number of accession (Table 1 and 2).

253 Table 1: Fungal isolates identified on untreated soil

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Isolates Identity Accession Scientific name

percentage number

T-1 96.3% NR_152267.1 Cladosporium oxysporum

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T-2 98.12% NR_153473.1 Nigrospora camelliae-sinensis

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T-3 98.44% NR_153480.1 Nigrospora hainanensis

T-4 99.81% NR_121232.1 Penicillium oxalicum

T-5 99%
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NR_168177.1 Curvularia xishuangbannaensis

T-6 99.44% NR_153480.1 Nigrospora hainanensis

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T-7 98.99% NR_168177.1 Curvularia xishuangbannaensis

T-8 99.6% NR_145243.1 Neopestalotiopsis rosae

T-9 99.62% NR_153480.1 Nigrospora hainanensis

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T-10 97.61% NR_137301.1 Trichoderma afarasin

T-11 100% NR_136119.1 Alternaria burnsii

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255 Table 2: Fungal isolates identified on treated soil

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Isolates Identity Accession Scientific name

percentage number
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T-1 98.56% NR_138223.1 Curvularia lunata

T-2 98.86% NR_153480.1 Nigrospora hainanensis

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T-3 96.82% NR_152503.1 Curvularia soli

T-4 99.43% NR_152503.1 Curvularia soli

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 T-5 98.98% NR_168177.1 Curvularia xishuangbannaensis

T-6 99.42% NR_153474.1 Nigrospora osmanthi

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256

257 3.3 Direct confrontation test

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258 Among the endophytic fungi isolated from untreated soil, 6 were directly confronted with S. rolfsii.

259 The different measurements in Table 3 present the percentages of inhibition of S. rolfsii mycelial

260 growth . It appears that neither isolate has any inhibition effect on Sclerotium rolfsii . Among all the

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261 isolates, only the genus Nigrospora had an average antagonistic activity on the mycelial growth of S.

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262 rolfsii with an inhibition rate of 47.5% and 45% (Fig. 4).

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264 Table 3: Percentage of inhibition of mycelial growth of S. rolfsii in confrontation with isolates after

10 days of incubation
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265

Antagonists DM T (cm) DM P (cm)) I (%)

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Nigrospora hainanensis 8 4.2 47.5

Nigrospora camelliae-sinensis 8 4.4 45

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266 DM T : Mean Diameter of the Witness DM P : Mean Diameter of the Pathogen

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268
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269

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276 Fig. 4. Direct confrontation test of Nigrospora fungal isolates against S. rolfsii

277
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278 3.4 Purification of secondary metabolites from Alternaria burnsii and Neopestalotiopsis rosae

279 Since these endophytic fungi are recognized as phytopathogens, it was necessary to study the effects

280 of their secreted mycotoxins on tomato plants and in the soil. Thus, the crude extract obtained from
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281 Alternaria burnsii (Fig. S1) was purified by silica gel chromatography with a Cyclohexane/Ethyl

282 Acetate (10-0/0-10) gradient elution to give a yellow-orange powdery compound of mass (220 mg).
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283 This compound was identified by mass spectrometry (Applied Biosystem QSTAR Pulsar I mass

284 spectrometer) with a HR-ESI-MS molecular ion peak m/z [M+H] +258.0528 (calc. for C14H10O5 +
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285 H + : 258.0529) as alternariol (Fig. 5) . The structure was determined by analysis of 1D and 2D NMR

286 spectra (Fig. S2) (Bruker 400 MHz) in agreement with literature data (Koch et al., 2005).
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287
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 H3C OH

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HO

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288 OH O

289 Fig. 5 : Structure of the alternariol mycotoxin produced by A. burnsii

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290

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291 Neopestalotiopsis crude extract rosae (Fig. S3) with a mass of 627 mg obtained under the elution

292 system Cyclohexane – Ethyl acetate according to the proportions (70:30) in silica gel gave a

293
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compound in the form of white crystals with a mass of 225 mg. Mass spectrum analysis of the

294 compound isolated allowed to highlight a high-resolution molecular ion peak [MH] - at m/z 395.3297
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295 . The molecular mass is therefore 396.3375 g/mol . The analyses of the 1 H proton spectrum 1D NMR

296 and the carbon 13C spectrum (DEPT-Q) as well as the 2D NMR (Fig. S4) showed that the compound

297 at m/z 395.3297 calculated for C 28 H 44 O corresponds to ergosterol.

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298
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H3C CH3

CH3
CH3
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CH3
H3C
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299 HO

300 Fig. 7 : Structure of ergosterol produced by N. rosae

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302 4. Discussion

303 The aim of this work was to study the influence of mycoparasites isolated from tomato from

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304 two different habitats. According to the literature, environmental conditions classify endophytic fungi

305 into three types namely asymptomatic endophytes that do not cause disease to the host plant during

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306 almost the entire duration of their life cycle inside the host plant (Sinclair and Cerkauskas, 1996).

307 Symbiotic endophytes, they are asymptomatic but form beneficial and mutual associations with the

308 host plant (Sinclair and Cerkauskas, 1996). And Latent pathogenic endophytes, these are true

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309 phytopathogens but are only expressed if the plant is in unfavorable or stressful conditions such as

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310 drought, insect attack or parasitic pressure from soil-borne pathogens (Sinclair and Cerkauskas,

311 1996). In our case study, the emphasis is on the parasitic pressure of tomato pathogens.

312
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The first part of this study consisted in studying the influence that arises during the parasitic

313 pressure of soil pathogens on tomato plants. This work led to a diversity of endophytic fungi isolated
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314 on both sides of the tomato leaves from the two types of experimental soil. Among the isolates, there

315 are asymptomatic, symbiotic and latent pathogenic endophytes. A total of 17 fungi were isolated from

316 tomato leaves. 11 came from untreated soils and 6 were isolated from heat-treated soils. This
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317 difference could be explained by the fact that part of the experimental soil was treated allowing a

318 considerable reduction in the parasitic load of soil pathogens. Indeed, several parameters can
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319 influence the growth of a crop. Among these parameters are the quality of the soil, in particular the

320 microbial communities that compose it and the quality of the endophytic microbial communities.
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321 Furthermore, Bradford et al ., 2008 showed that soil warming causes changes in microbial

322 communities, in the form of a reduction in microbial biomass.

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323 The second part of this work consisted in identifying macroscopically and microscopically the

324 fungal isolates from the cultural elements and the identification keys according to the literature. This

325 diversity grouped several genera of microscopic fungi including the genus Nigrospora, Curvularia,
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326 Pestalotiopsis, Penicillium, Alternaria, Trichoderma and Cladosporium. This work joins that of

327 Larran et al., 2001 who isolated a diversity of endophytic fungi from tomato leaves ( Solanum
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328 esculentum ) among which are the 7 genera of endophytic fungi. The molecular identification was

329 done on the basis of the amplification of the ITS regions of their ribosomal DNA and revealed the

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330 presence of several fungal species on the two culture habitats. The species of the genus Nigrospora

331 and Curvularia were the most frequently isolated among the fungal isolates. Furthermore, Alternaria

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332 species burnsii and Neopestalotiopsis rosea isolated from tomato leaves grown on untreated soil are

333 recognized as latent pathogens according to literature data. Their pathogenicity is only expressed

334 when the host plant is in abiotic stress conditions related to the soil. This explains this lack of

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335 greenness of tomato plants on this soil. Indeed, Alternaria is responsible for alternaria in the plant. In

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336 the Middle East and Asia, Alternaria burnsii is responsible for cumin blight ( Feng et al., 2021) .

337 Alternaria mycelium growth occurs at an optimal temperature between 18 and 25 °C. However, spore

338 infection and germination can occur in a wide temperature range between 4 and 35 °C (Chain, 2011)
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339 . During infection, Alternaria species produce host-specific and non-specific phytotoxins and

340 extracellular enzymes to destroy plant cell walls at the infection site, which plays a major role in
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341 pathogenicity against plants (Wu and Wu, 2019) . Due to their tolerance to many environmental

342 conditions, Alternaria can infect a range of products in various geographical areas, resulting in the
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343 spread of its mycotoxins (Louro et al., 2024). Fungi belonging to the genus Alternaria produce

344 more than 70 known mycotoxins belonging to three different structural groups: dibenzopyrone
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345 derivatives, perylene derivatives, and tetramic acid derivatives. (Pinto and Patriarca , 2017) . The

346 most studied mycotoxins of Alternaria are those containing benzopyrone groups, which include the

347 two main toxins: Alternariol (AOH) and methyl -Alternariol (AME) (Escrivá et al., 2017).
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348 Alternariol is often present in cereals, fruits, and fruit-based food products such as jams and juices

349 (Puvača et al., 2022). High levels of AOH have also been observed in legumes, nuts, and tomatoes
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350 (Solhaug et al., 2016). In the case of our study, alternariol was isolated from Alternaria burnsii . This

351 result confirms well the claims in the literature. As for Neopestalotiopsis rosae, it is the cause of leaf
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352 blight and crown rot on strawberries worldwide (Wu et al., 2020). In this work, the chemical study

353 of this fungus made it possible to identify ergosterol and its derivatives as the major compounds.

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354 Ergosterol is a compound belonging to sterols, like cholesterol. This molecule has low solubility in

355 oil and is easily crystallized, which represents a problem for its direct use (Park and Kim, 2020) .

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356 However, this molecule becomes active when combined with other compounds (Park and Kim,

357 2020) . According to a work carried out by Barajas - Aceves, 2000. The ergosterol content in the soil

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358 was independent of the presence or absence of pollutants. Thus, the results of the experiments showed

359 that ergosterol can be a useful indicator of fungal biomass in polluted soils and can be used to monitor

360 bioremediation. Recall that these two pathogenic fungi were isolated for the first time as pathogens

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361 of tomato crop in Ivory Coast. Ultimately, these results of this study show that pathogenic endophytic

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362 fungi originate from the soil, hence the stress state of tomato plants on untreated soil.

363 Acknowledgement

364
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The authors would like to thank the African Centre of Excellence on Climate Change, Biodiversity

365 and Sustainable Agriculture (CEA-CCBAD) which funded this research work.
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366

367 Conflict of interest

368 The authors have no financial conflicts of interest to declare.

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369 Conclusion
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370 This study highlighted the effect of soil treatment on the soil microbial community. This resulted in

371 a considerable reduction in the microorganisms contained in the soil and their activity. Seventeen
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372 fungi were isolated and identified first by microscopy and molecular biology. The identified fungi

373 were grouped into seven genera: Nigrospora, Penicillium, Curvularia, Colletrichum, Pestalotiopsis,
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374 Cladosporium, Alternaria. Eleven came from untreated soils and six came from heat-treated soils.

375 Among the fungi isolated from untreated soils, Neopestalotiopsis rosae and Alternaria burnsii were
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376 recognized as latent pathogens of tomato, hence the stress state of the plants. Heat treatment of the

377 soil could be a method of controlling plant phytopathogens.

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378 References

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383 (2), 165-172.

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384 Barajas - Aceves , M., 2000. Soil ergosterol, dimethyl sulphide reduction and microbial biomass along

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385 a Zn concentration gradient in soil from a mine spoil tip. Bull. Approximately. Contact. Toxicol. 64,

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399 Gardes, M., Bruns, TD, 1993. ITS primers with enhanced specificity for basidiomycetes application

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424 Puvača N, Avantaggiato G, Merkuri J, Vuković G, Bursić V, Cara M, 2022. Occurrence and

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431 Fungi in Grasses and Woody Plants: Systematics, Ecology, and Evolution. Chap.1, 329

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432 Solhaug A, Eriksen GS, Holme JA, 2016. Mechanisms of action and toxicity of the mycotoxin

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436 esculentum Mill.) under shelter and incidence of transplanting age on plant vigor against Pythium sp.

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438 White TB, Lee S., Taylor J., 1990. Amplification and direct sequencing of fungal ribosomal RNA
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439 Genes for phylogenetics. ResearchGate. 315-322.

440 Wu HC, Wu WS, 2019. Evaluation of virulence and pathogenicity of Alternaria patula on French
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441 marigold (Tagetes patula). Plant Pathol . 68(4), 678–688. https://doi.org/10.1111/ppa.12982

442 Wu HY, Tsai CY, Wu YM, Ariyawansa HA, Chung CL, Chung PC, 2020. First Report of
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443 Neopestalotiopsis rosae Causing Leaf Blight and Crown Rot on Strawberry in Taiwan. Plant Dis. doi:

444 10.1094/PDIS-05-20-1045-PDN. Epub ahead of print. PMID: 32976075.

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 Influence of soil mycoparasites on tomato cultivation (Solanum lycopersicum ) in the

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locality of Songon in Côte d’Ivoire.

Sagou Dominique a *, Pakora Gilles Alex a , Amari Ler-N'Ogn Dade George Elisée b

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a Laboratory of Biology and Health, UFR of Biosciences, Félix Houphouët-Boigny University,

22 BP 582 Abidjan 22, Ivory Coast,

b Biotechnology Laboratory, Agriculture and development of biological resources, UFR of

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Biosciences, Félix Houphouët-Boigny University, 22 BP 582 Abidjan 22, Ivory Coast,

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*Author corresponding to: Laboratory of Biology and Health, UFR of Biosciences, Félix

Houphouët-Boigny University, 22 BP 582 Abidjan 22, Ivory Coast

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(+225) 0747969858 [email protected]
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Fig. S1 Phylogenetic tree for species A. burnsii (isolate T14) from tomato leaves of Ivory Coast

species emitted using the analysis (ITS) generated with the MEGA 11 application .
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Fig. S2 proton spectrum 1 H NMR 1D of alternariol

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Fig. S3 : Phylogenetic tree for the species N. rosae (T8 isolate) from tomato leaves from

Ivory Coast species emitted using the analysis (ITS) generated with the MEGA 11 application
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Ergosterol mass spectrum

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Fig. S4 1D 1H-NMR proton spectrum of ergosterol

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