Surveys of cotton fields were conducted throughout the cotton-growing region of Côte d’Ivoire to collect diseased cotton plants. Plant fragments (roots and stems) were collected from specimens showing browning of the stem vessels, a hallmark symptom of cotton tracheomycosis. Sampling was performed under strict aseptic conditions, ensuring no direct contact between different samples. Each sample was placed in a large polyethylene bag, clearly labeled with pertinent information including sampling location (date, geographical coordinates and cotton variety) and subsequently stored in a cold until further analysis.

Symptomatic plant samples were aseptically sectioned into small stem fragments measuring approximately 0.5 to 1 cm in length. These fragments were immersed for 3 minutes in a 15% diluted sodium hypochlorite solution at 8˚C, followed by three successive rinses in sterile distilled water. Subsequently, the fragments were placed on Potato Dextrose Agar (PDA) medium. The PDA medium consisted of 20 g bacteriological agar, 20 g glucose and 20 g potato flakes dissolved in 1000 ml distilled water, sterillized by autoclaving at 121˚C for 30 minutes. Petri dishes of 90 mm diameter were incubated in the dark at 25˚C for 48 hours prior to subculturing. After 3 to 4 successive subcultures on PDA medium, pure fungal cultures were obtained and stored at 25˚C in the dark within an incubator.

Identification of phytopathogenic strains was primarily conducted through the examination of their macroscopic and microscopic characteristics [11]. The principal diagnostic features employed included:

Cultural characteristics (color, mycelia appearance, etc.)Hyphal types (septate)Presence of macroconidia, abundant microconidia in the false headPresence of terminal or intercalary chlamydosporesPhialide characteristics (monophialides)Phialide size (short)

Microscopic observations and descriptions of the colonies were performed on isolates cultured on PDA medium for 7 days at 25˚C.

Descriptions were based on observation using a light microscope equipped with an integrated camera. For this purpose, a fragment of the fungal colony was aseptically transferred using a sterile platinum loop onto a microscope slide, stained with methyl blue and covered with a coverslip. Observations were carried out under the light microscope at G × 40 magnifications [12]. Cultural features such as colony appearance and pigmentation were recorded.

For the collection of fungal isolates, it is essential to obtain single-spore cultures [13][14]. The serial dilution technique was used for this purpose. Five ml of sterile distilled water were added to the surface of each culture maintained on PDA medium in 5 cm diameter dishes. The surface of the saturated cultures was gently scraped using the tip of a pipette. The resulting suspension containing conidia and mycelium fragments was filtered through sterilized muslin cloth. The filtrate was collected in tubes, and a series of successive tenfold dilutions was performed up to 10⁻⁶. From each dilution, 100 µl of homogenized suspension was inoculated onto malt medium (composition) in 9 cm diameter Petri dishes (13 ml of medium per dish). After incubation for one to two days at 25˚C in the dark, unicellular conidia that were sufficiently isolated and had formed germ tubes, were identified under a magnifying glass and subcultured at a rate of one colony per dish, onto malt medium supplemented with acid and an antibiotic (250 mg/l citric acid, 50 mg/l chlorine tetracycline, 100 mg/l streptomycin). Three successive subcultures on this medium ensure the bacterial purity of the monospore culture while fungal purity was guaranteed by the monospore subculturing itself. Ninety-one (91) isolates were thus purified and monosporic.

Due to the variability in the characteristics of Fusariumoxysporum isolates according to their geographic and plant origin [15], it was considered pertinent to characterize a subset of this isolate collection. For this purpose, 24 isolates from different geographic regions were selected based on morphological characteristics (morphotypes) revealed on culture media for further analysis. These isolates were chosen to be representative of all the morphotypes observed in the collection of 91 isolates. The growth rate of the selected isolates was evaluated on three different culture media over a period of 10 days. The media used were as follows:

PDA (Potato Dextrose Agar) medium, consisting of 20 g of potato flakes, 20 g of glucose, 20 g of bacteriological agar, and 1 liter of distilled water. Before distributing the medium into 90 mm diameter Petri dishes under a fume hood, 0.25 g of citric acid was added to prevent bacterial growth.Czapeck-Dox Agar (CDA) medium, consisting of 3 g of NaNO3, 1 g of KH2PO4, 0.5 g of KCl, 0.5 g of MgSO4 7H2O, 0.01 g of FeSO4 7H2O, 30 g of sucrose, 20 g of bacteriological agar, and 1 liter of distilled water.Malt medium, the composition of which is 20 g of malt extract, 20 g of bacteriological agar and 1 liter of distilled water.

All isolates used for the growth rate evaluation were of the same age and were cultured on the same date. To standardize this, a fragment of purified mycelial colony was aseptically transferred using a sterile loop onto PDA medium in Petri dishes. The plates were incubated at 28˚C in the dark for 10 days (Figure 1).

After 10 days of incubation, mycelial discs were excised from the periphery of the culture (Figure 2) using a 4 mm diameter punch and aseptically transferred to the center of sterile Petri dishes containing the different solid culture media (PDA, CDA, MALT). Seven replicates were performed for each culture medium and for each isolate [16]. The diameter of the fungal colonies was measured daily along two perpendicular axes (Figure 3).

Figure 1. 10-day-old Fov isolate.

Figure 2. Cultures showing discs of mycelia taken.

Figure 3. Measurement of the diameter of a mycelia colony of Fov isolates.

Radial growth was calculated according to the formula of Sofi and collaborators [17].

Cr: Radial growth

Cn: Diameter observed on a given day (mm)

Cn_1: Colony diameter on day n − 1 (mm)

The spore concentration of Fusarium oxysporum f.sp. vasinfectum isolates was determined from 10-day-old cultures. Five ml of distilled water were added to each Petri dish, and the surface was gently scraped using a Pasteur pipette. A drop of the resulting suspension was then placed on a blade of Malassez. Spore counts for each culture medium were repeated three times by enumerating spores along the diagonals. The spore concentration of the isolates was calculated using the formula:

n is the average number of spores counted in the smallest square of the blade.

All data were recorded in Excel 2007 which was used to generate graphs and pie charts. The raw data collected during this study were analyzed using STATISTICA 7.1 software. A one-way analysis of variance (ANOVA) was performed for the parameters. When a significant difference was detected among means (P < 0.05), pairwise comparisons were performed using Fisher’s LSD tests at the 5% significance level.

For the cultural characterization of Fov isolates, the following criteria were considered: mycelia appearance and colony color on PDA culture medium. After isolation, isolates were subcultured on PDA medium, after the fourth subculture showed variability in mycelial appearance and colony pigmentation. Figure 4 shows the percentage of different mycelial colorations observed among the isolates post-isolation. Of all strains 37 isolates (41%) displayed a pink coloration, 34 isolates (37%) exhibited a purple coloration, 3 isolates were white, and 7 isolates showed a salmon coloration and 10 isolates presented lavender mycelial pigmentation (Figure 4). The analysis of the pivot table encompassing mycelium appearance parameters and colonies coloration allows the classification of the isolates into homogeneous groups. Based on the different characteristics assessed, ten (10) groups of isolates were identified (Table 1).

Figure 4. Percentage of different mycelia colorations of fungal isolates.

Table 1. Composition of the different groups and their characteristics.

To characterize Fov isolates, their mycelial growth on various culture media (PDA, MALT, and CZAPECK) and spore concentrations were evaluated using 24 representative isolates from the collection (Table 2).

Table 2. List of representative isolates from the collection.

The mean radial growth rates of Fov isolates were significantly greater on CDA and Malt medium compared to PDA medium (Table 3).

At day 8, the growth rates of Fov isolates on the three culture media ranged from 33.91 ± 3.35 to 86.00 ± 0.00 mm (Table 3). Specifically, on PDA medium, mycelial expansion varied between 37.75 ± 3.76 to 86.00 ± 0.00 mm; on MALT, it ranged from 35.66 ± 3.14 to 86.00 ± 0.00 mm. The growth rates of isolates on CDA medium ranged from 33.91 ± 3.35 to 86.00 ± 0.00 mm. Isolates AM 94 and AM 98 exhibited the highest mycelial growth across PDA, MALT, and CDA media. Statistical analysis revealed a highly significant difference between isolates (P < 0.0001). The maximum average growths are respectively 64.39 ± 14.58 mm on CDA, followed by 57.88 ± 14.04 mm on malt and 56.11 ± 11.90 mm on PDA medium. Statistical analysis revealed that, after 8 days of incubation, a highly significant difference (P < 0.0001) was observed among the different media. The radial growth of the fungus was found to vary significantly depending on the type of medium.

Table 3. Radial growth of Fov isolates on different culture media.

For each average,the values bearing the same letters (a,b,c,and d) in the same column are statistically identical to the5%threshold.

Fov isolates cultured on PDA, MALT, and CDA media showed different sporulation depending on the culture medium. Figure 5 shows some microscopic views of the spores of Fov

Figure 5. Microscopic views of spores of some Fov isolates.

The spore concentration of Fov isolates varied across all culture media from 0 spores/ml to 2.69 × 108 spores/ml (Figure 6).

Figure 6. Effect of different culture media on spore concentration of Fov isolates.

On PDA and MALT media, concentrations were 0 spores/ml to 2.69 × 108 spores/ml and 0 spores/ml to 1.18 × 108 spores/ml, respectively. On CDA medium, it was 0 to 2.05 × 107 spores/ml. Isolates AM 98, AM 90, AM 9, AM 94, and AM 61 produced fewer spores across all three-culture media (Table 4). The highest spore counts came from isolates AM 16 on PDA medium, AM 12 on MALT medium, and AM 105 on CDA medium.

Statistical analysis showed a highly significant difference between isolates (P < 0.001) and between culture media (P < 0.001).

PDA medium allowed the isolates to produce a large number of spores. This medium revealed greater variability in spore concentrations among Fov isolates, with up to fifteen (15) homogeneous groups. MALT and CDA media only presented eight (8) and seven (7), respectively (Table 4).

Table 4. Spore concentration of Fov isolates on different culture media.

For each average, the values bearing the same letters (a, b, c, and d) in the same column are statistically identical to the 5% threshold.