Fusarium and Sarocladium Species Associated with Rice Sheath Rot Disease in Sub-Saharan Africa

1. Introduction

Rice (Oryza sativa) holds significant economic importance in Africa, with Nigeria being one of the leading contributors to the continent’s global rice production share (4.2%), accounting for 24% [1]. Despite this, Nigeria remains the second-largest importer of rice worldwide, trailing only behind China. In 2018 alone, Nigeria imported approximately 3 million metric tons of milled rice, struggling to meet its demand deficit for the past decade [2]. Mali, a landlocked country in West Africa, ranks fifth among African nations in terms of rice production. Its rice production is being managed through irrigated systems connected to the Niger River. In Rwanda, the demand for rice is estimated at 145,000 tons per year, while national supply accounts for about 40%, creating a 60% deficit that is met through imports [93]. While the rice cultivation area is expanding in Africa, the average yields (2.35 tons/ha in 2021) are low when compared to Asia (4.95 tons/ha in 2021) [1]. Rice production is constrained by various factors including biotic stresses such as pests and diseases. Africa has recorded a steady and substantial increase in the incidence of rice diseases such as Rice Yellow Mottle Virus (RYMV), Rice blast (Pyricularia oryzae), Bacterial Leaf Blight (Xanthomonas oryzae pv oryzae), Bacterial Leaf Streak (Xanthomonas oryzae pv oryzicola), and Rice Stripe Necrosis Virus (RSNV) [3,4,5,6,7].

Rice sheath rot is an emerging disease worldwide [8]. Sarocladium oryzae, [9], formerly Acrocylindrium oryzae, was the first organism to be associated with rice sheath rot symptoms in Taiwan in 1922 [10] Sarocladium attenuatum was originally described as a distinct species causing rice sheath rot, was then considered a synonym of Sarocladium oryzae [11], but has recently been re-established as a separate species causing rice sheath rot in Taiwan [12]. These authors also described a third species that causes sheath rot symptoms on rice called S. sparsum, which is closely related to S. oryzae and S. attenuatum. Sarocladium has been associated with rice sheath rot in thirty-eight countries [13].

Besides Sarocladium species, Fusarium spp. have been associated with the rice sheath rot complex. These mainly comprise isolates in the F. fujikuroi species complex (FFSC) including F. proliferatum, F. verticillioides, F. incarnatum and F. fujikuroi [14,15,16,17,18,19]. In addition, various bacterial species cause rice sheath rot symptoms. The most important one is Pseudomonas fuscovaginae which is known to cause sheath brown rot of rice at high altitudes. In Africa, this bacterium has been reported in Burundi [8] [20] and Madagascar.

In West Africa, rice sheath rot has been reported in Cote D’ivoire, Gambia, Niger, Nigeria, and Senegal [13]. However, no causative fungal strains were isolated and no detailed scientific information was provided except for Nigeria [21] where S. attenuatum was first reported in Nigeria as one of the causes of grain discoloration on rice [21]. In addition, an inhibitory effect of S. oryzae on seed germination was later observed [22]. Most information pertaining to the occurrence of S. oryzae in Africa relating to stored, marketed and field seeds especially with respect to mycotoxigenic potentials was enumerated by [23].

Rice sheath rot can cause high yield losses of 20-80% [24,25,26]. Furthermore, an extensive survey of rice fields across West Africa enabled the identification of sheath rot symptoms in Mali and Nigeria (AfricaRice disease database). Although yield losses due to the sheath rot disease has not been estimated in Mali and Nigeria, a field survey conducted in 2011 and 2013 revealed high incidence and severity of the disease in Rwanda [27].

Mycotoxin contamination of cereal products poses a serious concern for animal and human health. Several studies have reported Fusarium species as the major producers of mycotoxins contaminating cereals including rice [28,29,30,31,32,33,34]. In the African region, previous studies have reported several mycotoxins being synthesized by Fusarium species isolated from rice as a serious health threat to producers and consumer [35,36,37,38,39,40].

The comprehensive information regarding the incidence and distribution of sheath rot disease is the aspect notably lacking. Additionally, there has been a notable absence of research examining the genetic, pathogenic, and toxigenic variability of pathogens associated with this disease in East and West Africa. Acquiring this crucial information will offer valuable insights for disease control and enhance management strategies for breeding programs. Therefore, this study aimed at identifying, characterizing, as well as assessing the genetic, pathogenic, and toxigenic diversity of the pathogens associated with rice sheath rot disease in Mali, Nigeria, and Rwanda.

2. Materials and Methods

2.1. Collection of Samples

Samples were collected from rice research programs and farmer’s fields in Mali, Nigeria, and Rwanda (Figure 1). Naturally infected whole rice plants with sheath rot symptoms having sheath browning, necrosis, grain emptiness and rotting as indicated in Figure 2 were collected. Samples from farmer’s field were collected at the office of the rural development, Selingue village near Bamako, Mali and from two fields located at Ibadan, Oyo State and Katcha near Badeggi, Niger State in Nigeria during the 2017 rice growing season. Samples were randomly collected 25 m apart at each location. The samples collected were conserved in dry paper bags, while hands were disinfected with 70% alcohol after each sampling. Samples were later stored in dry bags in the refrigerator at 4°C in the laboratory. Isolates earlier collected from Rwanda in 2011 and 2013 were also included in this study. Agro-climatological details of the selected three countries with their various agroecologies are presented in Table 1. Isolation and identification of pathogens was carried out at the Phytopathology laboratory of Ghent University, Belgium.

2.1.1. Isolation and Purification of Sheath Rot-Associated Isolates

Infected sheath and seed samples showing symptoms of sheath rot were surface - sterilized in 2% sodium hypochlorite for two minutes and then rinsed thrice in sterile distilled water. They were drained using sterile paper towels, and thereafter cut into small pieces of about 0.5 cm² and plated on 90 mm diameter Petri dishes containing Potato Dextrose Agar (PDA). The cultured Petri dishes were incubated at 28°C in darkness for 7-14 days. Cultures were further purified by plating on fresh PDA.

2.1.2. Identification of Pathogens

Identification of fungal pathogens was done based on their typical structure and basic characters as described by Barnett and Hunter (1972). The incidence and diversity of fungi was observed and recorded. After 5 days, all plates were examined under a compound microscope for the presence of several fungal pathogens. Conidia of these fungi were mounted on glass slides in water and examined under a compound microscope for identification at genus level. For Fusarium species, pure cultures were plated and stored on PDA slants at room temperature and maintained at -80°C with 40% glycerol. A similar method of storage was used for Sarocladium species except for the use of 20% glycerol.

2.2. Molecular Characterization of Isolates

2.2.1. DNA extraction, amplification, and sequencing

Fungal isolates were grown on potato dextrose broth (PDB) at 28 °C for seven days. Mycelia mats were harvested by filtration, dried by blotting using sterile paper towels, frozen in liquid nitrogen, and pulverized using a tissue lyser (MM400, Retsch GmbH, Haan, Germany).

Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega). Quantification and purity were determined using Nanodrop 3000 (Thermo Scientific, Asheville, NC, USA) and diluted to a concentration of 20 ng µL-1.

Fusarium isolates were further identified by amplifying the Translation Elongation Factor (EF-1α), using primer pair TEF-1-F (5’-ATG GGT AAG GAA GAC AAG AC-3’) and TEF-2-R (5’-GGA AGT ACC AGT GAT CAT GTT-3’) [41]. PCR reactions were done in 25 µL of a solution consisting of 2 µL genomic DNA (100 ng µL-1), 5 µL PCR buffer (5x; Promega), 5 µL Q solution (Qiagen), 0.5 µL dNTPs (10 mM; Fermentas GmbH), 1.75 µL of each primer (10 µM), 0.15 µL Taq DNA polymerase (5 units µL−1; Fermentas GmbH) and 8.85 µL ultrapure sterile water. Amplification was performed with initial denaturation step at 94 °C for 1 min, followed by 35 cycles of denaturation at 94°C for 30 s, primer annealing at 53°C for 45 s and extension at 72 °C for 1 min. Cycling ended with a final extension step at 72 °C for 5 min [41]. The amplicons were separated by horizontal electrophoresis using 1.5 % agarose gels in TAE-buffer at 100 V for 25 min and visualized by ethidium bromide staining on a UV trans illuminator. Amplified products were purified with exosap and sequenced by LGC Genomics GmbH (Berlin, Germany) using Sanger sequencing.

For Sarocladium isolates, two genomic regions, the Internal Transcribed Spacer (ITS) and Actin, were amplified and sequenced. For the ITS region, primers ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’- TCC TCC GCT TAT TGA TAT GC -3’) were used (White et al., 1990). The actin region was amplified using ACT1 (5’- TGG GAC GAT ATG GAG AAG ATC TGG CA -3’) and ACT4 (5’- TCG TCG TAT TCT TGC TTG GAG ATC CAC AT-3’) [42].

For both primer pairs, PCR reactions were done in 25 µL of a solution consisting of 2 µL genomic DNA (100 ng µL-1), 5 µL PCR buffer (5x; Promega), 0.5 µL dNTPs (10 mM; Fermentas GmbH), 1.75 µL of each primer (10 µM), 0.15µL Taq DNA polymerase (5 units µL−1, Fermentas GmbH) and 13.85µL ultrapure sterile water. Amplification was performed using a Flexcycler PCR Thermal Cycler (Analytik Jena). For ITS amplification, the thermal profile consisted of an initial denaturation step at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min and extension at 72 °C for 1 min. Cycling ended with a final extension step at 72 °C for 10 min [43]. ACT fragments were amplified using an initial denaturation step at 94°C for 1 min, followed by 39 cycles of denaturation at 94 °C for 45 sec, primer annealing at 59 °C for 30 sec and extension at 72 °C for 30 sec. Cycling ended with a final extension step at 72 °C for 8 min [42].

2.2.2. Phylogenetic Analysis

The nucleotide sequences generated by the forward and reverse primers were used to obtain consensus sequences after editing via BioEdit version 7.2.5. [44]. From each duplicate identical sequence, a representative sequence from each identical set of sequence was compared to other sequences available at GenBank. Sequences were first aligned via muscle alignment in Mega V.11. [45] after which a maximum-likelihood tree was constructed based on the matrix of pairwise distances obtained using the General Time Reversible (GTR) Model. Reference sequences of Fusarium (Table 5) and Sarocladium (Table 6) species representing the three countries were used for phylogenetic analysis. For Fusarium, Phylogenetic tree was constructed based on maximum-likelihood inferred from partial EF-1α sequences of four Fusarium species complexes using IQ-Tree with GTR + G + I model and annotated using iTol software [46]. Cylindrocarpon sp. AC2011 strain CPC 13531 was used as an outgroup. However, for Sarocladium characterization, concatenated alignments of ITS and ACT region were performed after which a single phylogenetic tree was generated. To root the tree, Sarocladium zeae strain CBS 800.69 was used as an outgroup.

2.3. Pathogenicity Assay

Location of the isolates, and genetic groupings were used to select a subset of twenty-nine isolates for pathogenicity studies on rice plants. For pathogenicity tests with Fusarium species, representative isolates from the four Fusarium species complexes recorded in all the locations (FIESC, FFSC, FOSC, FSAMSC) were used for rice inoculation. To evaluate the pathogenicity of Sarocladium species, thirteen isolates comprising S. attenuatum (3), S. oryzae (6), and S. sparsum (4) were used. Indica rice cultivar (FARO 44) released by Africa Rice Center was used for the assay.

Inoculum was prepared according to the standard grain inoculum technique [47]. Briefly, rice grains were soaked in water for 60 min, excess water was removed, and the grains were autoclaved twice on two different days. For 4 g of rice grains, 1 plug (diameter = 5 mm) from the edge of a 14-day-old fungal colony was added together with 1 ml of sterile distilled water. Every two days, the grain inoculum was shaken to prevent the formation of clumps. After 10 days of incubation at 28°C, the inoculum was fully colonized.

The rice seeds were dehulled and surface sterilized in 2% sodium hypochlorite solution for 25 min, rinsed five times in sterile distilled water, and placed in Petri dishes containing sterile moistened filter papers (Whatman, grade 3). Following seedling emergence, six seedlings were transplanted into perforated plastic trays (22 x 15 x 6 cm) containing potting soil (Structural; Snebbout, Kaprijke, Belgium). Plants were watered daily, fertilized weekly with 0.2% iron sulphate and 0.1% ammonium sulphate, and maintained in a growth chamber (28°C, 60% relative humidity). Six-week-old plants were used for the inoculation.

One fully colonized fungal grain was introduced in the junction point between the sheath of the second youngest plant leaf and the stem. Inoculation points were covered with moist cotton wool and wrapped with parafilm to maintain humidity. High humidity was maintained for 24 h post inoculation by incubating in a controlled room at 28°C day and night, 16/12 light regime, and 85 % relative humidity. Subsequently, incubation was maintained at 65 % relative humidity, temperature, and light regime as above for 2-10 days. The disease development was evaluated eight days after inoculation by measuring the lesion length on the flag leaf sheath. Each treatment was replicated thrice while the experiment was conducted once.

2.4. Statistical Analysis

Lesion length was used as a measure of isolate virulence on rice plants. Since the conditions of normality were not met, a non-parametric analysis was carried out. Lesion length of the infected sheath were quantified using Kruskal-Wally’s Rank Sum test followed by a post hoc Mann-Whitney test. Statistical significance was defined as P= 0.05.

2.5. Mycotoxin analysis

2.5.1. Culture Preparation

Pure cultures of seventy-seven identified isolates (Fusarium - 46, Sarocladium – 31) were sub-cultured on Petri dishes with PDA. The medium was poured into 90 mm Petri dishes. Two mm of clean and pure cultured isolates were sub-cultured on fresh PDA plates and incubated at 25 °C for 3 weeks. Each isolate was grown in triplicates.

2.5.2. Reagents and Standards

Ethyl acetate and dichloromethane (DCM) were purchased from (Thermofisher scientific, Merelbeke, Belgium). Analytical grade formic acid (100%) and ammonium acetate were from (Merck, Darmstadt, Germany). Purified water was from the Arium pro VF system (Millipore, Belgium). LC-MS grade acetic acid and methanol were from (Biosolve, Valkenswaard, the Netherlands).

Certified mycotoxin standard solutions, more specifically aflatoxin mix (AFB1, AFB2, AFG1 and AFG2), deoxynivalenol (DON), fumonisin mix (FB1 and FB2), nivalenol (NIV), neosolaniol (NEO), OTA, T2, HT2, 3-acetyldeoxynivalenol (3-ADON), diacetoxyscirpenol (DAS), 15- acetyldeoxynivalenol (15-ADON), fusarenon-X (F-X), sterigmatocystin (STC), zearalenone (ZEN) and deepoxy-deoxynivalenol (DOM) were purchased from Biopure (RomerLabs, Getzersdorf, Austria). Fumonisin B3 (FB3) and enniatin B (ENN B) were obtained from Fermentek (Jerusalem, Israel). Alternariol (AOH), alternariol monomethylether (AME), were purchased from Sigma-Aldrich (Bornem, Belgium) and roquefortine (ROQ-C) from Alexis Biochemicals (Enzo Life Sciences BVBA, Zandhoven, Belgium).

Working solutions were prepared by diluting the stock solutions in methanol and stored at -20°C. A standard mixture consisting of the above mycotoxins (without DOM) in a concentration range between 0.5 ng/µl and 40 ng/µl was prepared as well and stored at -20°C.

Mobile phase A (94% water, 5% methanol, 1% acetic acid and 5mM ammonium acetate) and mobile phase B (97% methanol, 2% water, 1% acetic acid and 5mM ammonium acetate) were prepared.

2.5.3. Sample Preparation and Extraction

The extraction process started with the preparation of the quality control samples. Briefly, three plugs each of blank agar (uninoculated) were removed and placed into each of the three 50 ml Falcon tubes (spike 1, spike 2, and blank) and were macerated into pieces using a sterile scalpel blade. Then 50 µL DOM internal standard (50 ng/µl) was added into each tube, after which 25 µL and 100 µL of the standard mixture were added to spike 1 and spike 2, respectively. The mixtures (spikes and blank) were left in the dark for 15 minutes.

Following the control sample preparation, Fusarium mycotoxins were extracted from pure cultures of different isolates by using a sterile 9 mm cork borer and scalpel to take three plugs (2 sides + centre). The plugs were transferred into 50 ml Falcon tubes and macerated into pieces using a sterile scalpel blade. Then 50 µL DOM internal standard (50 ng/µl) was added into each tube and left in the dark for 15 minutes. The samples together with the quality control samples were extracted by adding ethyl acetate + 1% formic acid. The content was agitated gently on a vertical shaker for 20 min and centrifuged at 3000 g for 15 min. Then a folded filter paper (VWR International, Zaventem, Belgium) moistened with ethyl acetate + 1% formic was placed on a new extraction tube to collect the upper layer of the filtrate. Thereafter, 5 ml of dichloromethane (DCM) was added to each of the samples. The mixtures were agitated on a vertical shaker for 20 min and centrifuged at 3000 g for 15 min. Following centrifugation, the bottom layer (DCM phase) was collected in the same Falcon tube with the same filter paper. The filtrates were evaporated to dryness at 40 °C under a gentle nitrogen stream. The dissolved residue was reconstituted in 200 µl injection solvent (60% mobile phase A and 40% mobile phase B), well vortexed, and ultra centrifuged for 5 min at 10000 rpm. Finally, 100 µl of the filtrates were transferred into HPLC vials for LC-MS/MS analysis.

2.5.4. Multi-metabolite analysis (LC-MS/MS)

The samples were analysed using a Quattro Premier XE triple quadrupole mass spectrometer coupled with a Waters Acquity UPLC system (Waters, Milford, MA, USA).

Liquid chromatography conditions and MS parameters were followed as described by [48].The analytical column used was a symmetry C18, 5 µm, 2.1 x 150 mm with a guard column of the same material (3.5 µm, 10 mm x 2.1 mm) (Waters, Zellik, Belgium) kept at room temperature. The injection volume was 10 µl. Capillary voltage was set at 3.2 kV with a source block temperature and desolvation temperature of 120 and 400 °C respectively. Data processing was done using the Masslynx and Quanlynx software.

3. Results

3.1. Sampling and Isolation

Information on Fusarium isolates obtained from diseased rice plants in Nigeria, Mali and Rwanda is presented in Table 2. Of the 46 Fusarium isolates evaluated in this study, nine isolates were obtained from seeds, while 37 isolates originated from the rice sheath. The highest number was obtained from Rwanda (24 isolates), followed by Nigeria (15 isolates), while Mali (seven isolates) had the least.

Information on Sarocladium isolates obtained from diseased rice plants in Nigeria, Mali and Rwanda is given in Table 3. Out of the 24 Sarocladium isolates obtained, four were from seeds and 20 from the rice sheath. The highest number of isolates were obtained from Nigeria (nine from Katcha, seven from Ibadan), six isolates were from Rwanda, and only two from Mali (Table 3).

Altogether, seventy isolates comprising Sarocladium species (24) and Fusarium species (46) were obtained. Four ITS characterized CBS Sarocladium species were added, and we sequenced their actin region in this study (Table 2).

3.2. Phylogenetic Analysis of Fusarium and Sarocladium-like spp.

3.2.1. Fusarium Species

Partial sequences of TEF-1a gene revealed the identity of all the 46 Fusarium isolates used. Similarities to DNA sequences in the Fusaroid-ID and GenBank database ranged from 99–100%. Members of four species complexes were identified: F. fujikuroi species complex (FFSC – 48%), F. incarnatum-equiseti species complex (FIESC – 35%), F. oxysporum species complex (FOSC – 13%) and F. sambucinum species complex (FSAMSC – 4%) (Table 2). The phylogenetic analysis of the 46 Fusarium isolated is presented in Figure 3. The origin and Genbank accession numbers of the reference isolates used in given in Table A1.

FFSC isolates from Rwanda clustered with F. andiyazi (7 isolates), F. madaense (6 isolates) and F. casha (1 isolate). FFSC isolates from Mali were identified as F. nygamai (5 isolates) and F. annulatum (1 isolate), while in Nigeria, 2 F. marum isolates were found.

FIESC isolates were found in Nigeria and Rwanda and belong to five different species (sulawesiense, pernambucatum, tanahbumbuense, hainanense, and incarnatum) previously classified from rice, cereals, insects, and human samples [31], [35], [36], [50,51,52,53] (Figure 3). Eight of our FIESC isolates were nested within the F. sulawesiense clade including 7 isolates from Ibadan and an isolate from Katcha. One isolate from Ibadan clustered with members of F. pernambucatum, two isolates from Katcha nested within the F. tanahbumbuense clade, and two isolates from Ibadan nested within the F. hainanense group. Three isolates from Rwanda were found in F. incarnatum (Figure 3). None of our isolates clustered with the F. equiseti species clade (Figure 3).

The FOSC was found in Rwanda and Mali with members belonging to F. callistephi (5 isolates from Rwanda), and F. triseptum (1 isolate from Mali). Two isolates belonging to F. acasia-mearnsii in the FSAMSC were obtained from Rwanda.

3.2.2. Sarocladium Species

Thirty-one S. oryzae-like isolates were used for phylogenetic analysis (Nigeria=16, Mali=2, Rwanda=6, reference isolates =7). Partial sequences and concatenation of both ITS and ACT regions showed the identities of all the isolates. They were further subjected to BLASTn comparison with isolates in GenBank. Results revealed that all the 31 isolates had 98 -100% identity with Sarocladium species. A concatenated tree, in which reference sequences from GenBank were included (see Table A2), clearly delineated the Sarocladium isolates into three distinct phylogenetic groups with high bootstrap values (Figure 4). Most isolates from Nigeria (11 of 16), one isolate from Mali (SEMA0013A) and one isolate from Rwanda (RFRG2) clustered together with reference isolates CBS 180.74 from India, CBS 361.75 from Kenya, and CBS120.817 from Panama and were identified as S. oryzae. The second Mali isolate (SEMA0029) and five of the six Rwandan isolates clustered with reference isolates CBS 101.61 from Mexico and CBS 399.73 from India and belong to the S. attenuatum lineage. Five isolates from Nigeria clustered with reference isolate CBS 414.81 from Nigeria and the S. sparsum isolate 18042 from Taiwan (Figure 4). They mostly occurred in Ibadan-Nigeria (DS) except for an isolate (BDNG0025) found on infected seed in Katcha (SGS). Finding a substantial number of this group in Ibadan was not strange because it clustered with a reference isolate CBS 414.81 of Ibadan origin, collected, and reported [21], and later deposited into the GenBank [42], and recently reclassified as S. sparsum.

3.3. Pathogenicity Testing

Representative isolates from the dominant Fusarium groups recorded in Nigeria, Rwanda and Mali were used for pathogenicity testing on the FARO 44 rice variety. All the four Fusarium species complexes could induce rice sheath rot symptoms on the rice cultivar, but the degree of virulence of all the Fusarium species tested showed significant variations on the rice cultivar (Figure 5). Specifically, one of the two isolates of F. marum IBNGF0016 from Ibadan in Nigeria had the highest disease severity on FARO 44 followed by the second F. marum isolate IBNGF0012 and an FIESC isolate F. sulawesiense BDNGF0002 from Katcha in Nigeria. On the contrary, F. nygamai originated from Mali and one of the FIESC isolate F. tanahbumbuense BDNGF0001 from Ibadan-Nigeria were the least virulent isolates.

For the pathogenicity tests with Sarocladium species, disease evaluation at 8 days post inoculation (DPI) showed that all the thirteen isolates tested could induce typical sheath rot symptoms on FARO 44 albeit with varying disease development levels (Figure 6). Isolates affiliated with S. sparsum, all of Nigeria origin, were the most aggressive (p < 0.05). In contrast, isolates affiliated with S. oryzae and S. attenuatum were less virulent (Figure 6). S. oryzae isolate IBNG0011 from Nigeria is the most aggressive among the group while SEMA0013A from Mali appears to be the least virulent.

3.4. Mycotoxin Profiling (In vitro)

Forty-six Fusarium and thirty-one Sarocladium species obtained from rice with sheath rot symptoms were investigated for multi-mycotoxin production using LC-MS/MS . None of the Sarocladium isolates produced mycotoxins. Our results revealed that the Fusarium species were able to produce eight mycotoxins. The most detected mycotoxins include type A (DAS and NEO) and B (NIV and FUS-X) trichothecenes which were produced by FIESC isolates obtained across the two regions of Nigeria albeit at different concentrations (Table 4). Furthermore, zearalenone (ZEN) at a very high concentration of 26,173 and 32,529 µg/kg was detected in the two Ibadan-Nigerian F. hainanense isolates IBNGF0005 and IBNGF0003, respectively. Two Rwandan isolates F. madaense RFRM18 and F. acasiae-mearnsii RFRM19 produced ZEN at the concentration of 1349 and 329 µg/kg, respectively. The predominant mycotoxins detected from Malian F. nygamai isolates were fumonisins (FB1, FB2, and FB3), which occurred at a very high concentration. FB1 concentrations ranged from 53,118 – 141,102 µg/kg followed by FB2 ranging from 3,391 - 5,122 µg/kg while FB3 occurred at a lower concentration which ranged between 355 – 692 µg/kg. Besides F. nygamai, F. annulatum SEMAF17-225A from Mali produced DAS, FUS-X, NIV, and NEO at different concentrations (Table 4).

5. Conclusion

In conclusion, our study showed that diversity occurs within the East (Rwanda) and West African (Mali and Nigeria) isolates of Fusarium and Sarocladium. Notable correlation was observed between the virulent group and collection area. The toxigenic profile of both pathogens was elucidated, and we found that most Fusarium isolates from Nigeria and Mali were able to produce one or more mycotoxins. In contrast, only two out of 24 isolates from Rwanda were able to produce mycotoxins. Further investigations with a broader geographic scope and a larger collection of samples are necessary to examine pathogenic variability and the population genetic structure. Meanwhile, based on the distinct groups of isolates from different regions, breeders in various agroecological regions should take note of the variations in virulence. This information can serve as a basis for selecting strains useful for identification and selecting effective sources of resistance for local rice breeding programs.

Moreover, additional studies are required to determine whether Sarocladium and Fusarium individually contribute to the observed symptoms in the field or if there is an interplay between both pathogens in the rice sheath rot complex. To the best of our knowledge, this research provides the first comprehensive dataset on the distribution, genetics, pathogenicity, and toxigenic profile of Fusarium species associated with rice sheath rot disease in sub-Saharan Africa.