1. Introduction

AJPS

American Journal of Plant Sciences

2158-2742

Scientific Research Publishing

10.4236/ajps.2023.143020

AJPS-123951

Articles

Biomedical&Life Sciences

Morphological and Molecular Identification of Fungi Associated with Sesame Diseased Plants of the Three Agroclimatic Zones of Burkina Faso

Wendolian

Romain Soalla

¹Pawindé

Elisabeth Zida

¹Bouma

James Neya

¹Kadidia

Koita

Laboratoire Biosciences, Université Joseph Ki-ZERBO, Ouagadougou, Burkina Faso

Laboratoire de Phytopathologie, Institut de l’Environnement et de Recherches Agricoles (INERA), Ouagadougou, Burkina Faso

23032023

14032903077, January 202326, March 2023 29, March 2023

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Sesame is Burkina Faso’s second essential agricultural export after cotton. It’s consequently a supply of income for producers and foreign exchange for the country. However, sesame production is characterized by low average yields of about 538 kg·ha ^-1 at the farmer’s field as compared to the potential yield of the improved varieties (1500 - 2000 kg·ha ^-1). Fungal diseases are some of the major constraints to sesame production in Burkina Faso. The present study contributes to the development of means to control pathogenic fungi of this crop, which are responsible for significant losses. The objective is to identify the fungi associated with diseased sesame plant samples. To this end, 149 samples of diseased sesame plants were collected from different production sites located in three agro-climatic zones of the country. The analysis of the samples according to the blotting paper method, based on the morpho logical characteristics of the fungi, allowed the identification of 18 genera with prevalence rates from 2.68% to 97.98%. The most frequently identified genera were Macrophomina (97.98%), Cercospora (86.57%), Fusarium (85.23%), Phoma (62.41%) and Colletotrichum (61.07%). The results also showed a variable distribution of fungi according to the agro-climatic zone with the predominance of Macrophomina in all three zones. Molecular identification by DNA sequencing of 120 isolates belonging to the different fungi detected allowed the identification of 25 species of which the most representative were Macrophomina phaseolina, Cercospora sesami, Corynespora cassiicola, Alternaria simsimi, Alternaria porri, Fusarium oxysporum, F. fujikuroi, F. equiseti, Colletotrichum capsici, and C. gloesporiodes. The present study showed that diseased sesame plants collected from different production sites in Burkina Faso housed several species of fungi. The fungi presence in diseased plants indicates the need to inform and raise the stakeholders’ awareness about the phytosanitary problems of sesame, but also to develop effective and appropriate control methods against these crop pathogens in Burkina Faso.

Burkina Faso Fungi Molecular Identification Morphological Identification Sesame

1. Introduction

Sesame (Sesamum indicum L.) is an important annual legume cultivated throughout the world and mainly in the tropics [1] . Due to the rate of 50% edible oil content of its seeds [2] , sesame is considered the queen of oil crops. Sesame oil is appreciated in Africa, Asia and even worldwide for its high quality and stability, [3] as well as for its therapeutic virtues [4] . An Iranian study on the benefits of sesame oil focused on metabolic syndrome (MetS), also known as insulin resistance. MetS is defined by the World Health Organization (WHO) as a group of symptoms including obesity, type 2 diabetes, dyslipidemia and hypertension that together increase the risk of coronary heart disease, stroke and other serious health problems. This study found the beneficial effects of sesame oil enriched with vitamin E supplementation on cardiometabolic factors in people with MetS [5] .

The main sesame-producing countries in the world are Sudan (1,525,104 t), Myanmar (740,000 t), Tanzania (710,000 t), India (658,000 t) and Nigeria (490,000 t) [6] . With about 63% of world production, Africa is the leading sesame-producing continent, followed by America [6] [7] . Burkina Faso is the second largest producer in the West African sub-region after Nigeria.

Sesame is a crop generally adapted to the dry climate of the world’s tropical regions, which can also be cultivated in humid zones of tropical and subtropical areas [8] . It is produced throughout the three agroclimatic zones of Burkina Faso by mostly poor farmers whose production constitutes an important source of income. Sesame is a cash crop of which extra than 80% of the production is for sale and export particularly [9] . It is the second most important agricultural export after cotton and is a source of foreign exchange for the country. Sesame production has become a tool in fighting against poverty because it allows producers to increase and diversify their income sources. Sesame yield in Burkina Faso is improved in recent years but remains, at around 723 kg·ha⁻¹ [6] compared to the potential yield (1500 to 2000 kg·ha⁻¹) of improved varieties popularized in the country. These low yields are the result of poor access to inputs by the smallholder farmers, irregular rainfall, and biotic constraints, notably insect attacks and diseases caused by microorganisms that seriously constrain sesame production in Burkina Faso. Sesame cultivation is subject to fungal diseases that occur at all stages of the plant’s growth. These diseases generally manifest in the field as leaf blights and necrosis, stem and root rot, wilting and plant mortality. Very little or no work has been done on the formal identification of fungal diseases of sesame in Burkina Faso. However, the main symptoms observed in the field could be attributed to charcoal rot of Macrophomina phaseolina [10] [11] , Cercospora leaf spot of Cercospora sesami [12] and fusarium wilt of Fusarium oxysporum [13] .

According to Langham et al. [14] , the major diseases of sesame are downy mildew, leaf spots due to Cercospora and Alternaria, and root and stem rots caused mainly by the soil-borne fungi of Fusarium, Macrophomina, and Phytophthora genera. In addition to these genera, Colletotrichum and Corynespora are also present, with some species attacking all parts of the sesame plant. The effective management of such diseases will contribute to yield increase and the country’s economy. Accurate identification of the fungi responsible for the main fungal diseases is a prerequisite for the development of efficient control strategies against this important sesame constraint.

The present study aims to identify at morphological and molecular levels, the fungi associated with diseased sesame plants from the three agroclimatic zones of Burkina Faso.

2. Methodology2.1. Collection of Diseased Sesame Plant Samples

Samples of diseased sesame plants were collected from the three agroclimatic zones of Burkina Faso during the 2017, 2018 and 2019 rainy seasons. These samples consisted of whole plants or organs showing symptoms of necrosis, decay, wilting, blight, or partial and total mortality were collected from sesame fields near national roads and then placed in Craft paper bags. The bags were labelled with the name and geographic coordinates of the collection site. Figure 1 below shows the collection site in the three agroclimatic zones.

2.2. Morphological Identification of Fungi

The collected samples were treated separately according to plant organs. The diseased plants were washed with tap water to remove soil residues and other inert particles. The different organs were cut into small symptom-bearing particles and disinfected with 70% ethanol for 45 seconds. These plant fragments were then placed in Petri dishes (90 mm Ø) previously lined with three layers of blotting paper soaked in sterile distilled water. The dishes were then incubated for 5 - 7 days in a chamber at a temperature of 22˚C ± 3 and an alternating cycle of 12 hours per day of darkness and near-ultraviolet light.

At the end of the incubation, the Petri dishes were observed under a stereo-microscope and a microscope to identify the fungi that had grown on the plant fragments. The identification was done based on macroscopic (color and aspect

of mycelium) and microscopic (shape and structure of conidia and mycelium) characteristics as described in the identification manual of [15] . For most fungi, identification was limited to the genus level.

The identified fungi were reported on an identification sheet according to the infected organ. The prevalence rates of the different fungi associated with diseased plants were calculated according to the formula below:

P i = N i N × 100

with Pi = Prevalence of fungus i; Ni = Number of samples of diseased sesame plants infected with fungus i; N: Total number of samples of diseased sesame plants examined

The prevalence rates according to the plant organ from which the fungi were detected were also calculated by the formula below:

P i o = N o i N o × 100

where Pio is the prevalence of fungus i detected on plant organ o (leaf, stem, root or capsule); Noi the number of samples of organ o infected by fungus i and No, the total number of organ o samples examined.

2.3. Isolation of Fungi

Morphologically identified fungi were isolated in Eppendorf tubes containing sterile distilled water to form isolates and stored in the refrigerator at 4˚C as conidial or mycelial suspension. One drop of each suspension was spread on a Petri dish containing agar medium and incubated at laboratory conditions (25˚C ± 3) for 12 to 24 hours. Five germinating spores were then isolated and transferred to new Petri dishes containing Potato Dextrose Agar (PDA) for one spore per dish and placed in the incubation chamber for growth under the same conditions described above. The resulting pure single-spore isolates were stored for further study.

2.4. DNA Extraction

Single-spore isolates were grown in Potato Dextrose Broth (PDB) liquid medium. A 4-mm diameter mycelial explant of each isolate from a 7-day-old culture on PDA medium was aseptically collected and deposited into a PDB medium contained in a 250-ml Erlenmeyer flask. The inoculated media were incubated at laboratory room temperature (25˚C ± 3), with shaking at the speed of 100 oscillations per minute, for two to five days. At the end of the incubation, the Erlenmeyer contents were filtered with a vacuum pump and the mycelium was collected in an Eppendorf tube and dried in an oven at 27˚C for 48 to 76 hours.

The DNA extraction concerned 120 isolates of fungi including 20 belonging to the genus Macrophomina, 30 to the genus Fusarium, 20 to the genus Cercospora, 11 to the genus Alternaria, 10 to the genus Colletotrichum, 6 to the genus Phoma, 5 to the genus Curvularia and 18 to other genera including Nigrospora (2), Cladosporium (2), Exserohilum (1), Pestalotia (1), Phomopsis (1), Rhizoctonia (1), Melanospora (1), Myrothecium (1) and Botryodiplodia (1), Aspergillus (2) and the unknowns (5).

Mycelium samples contained in 2 ml Eppendorf tubes were then ground using Tissue Lyser II and subjected to DNA extraction following the Cetyl-Trimethyl-Ammonium Bromide (CTAB) method of Ford et al. [15] with some modifications. A volume of 600 µl of CTAB solution (1.4 M NaCl; 2% CTAB (w/v); 0.1 M Tris-Base pH8; 0.02 M EDTA pH8; 0.2 B-Mercaptoethanol (v/v)) was added to 75 - 100 mg of conidial powder and incubated at 65˚C for 10 minutes. Then 450 µl of chloroform: isoamyl alcohol (49:1) was added to the tube containing the sample, vortexed gently, and centrifuged at 13,000 g for 5 minutes at 25˚C. The same process with lsoamyl alcohol was repeated with the top part of the first step. The top part of the second step was transferred into a 1.5 ml tube to which 0.7 volume of isopropanol solution was added to precipitate the DNA at −20˚C for 30 minutes. After centrifugation at 13,000 g for 10 minutes, the liquid in the tube was removed and the pellet was rinsed with 500 µl of Ethanol at 70% by centrifugation for 3 minutes at 13,000 g at 25˚C. The rinsing process was repeated a 2nd time. The DNA contained in the tubes was dissolved by adding 50 µl of sterile distilled water.

DNA concentrations were then determined using the NanoDrop 2000 spectrophotometer.

2.5. Amplification of DNA from Isolates

The DNA samples were amplified by PCR, targeting regions 1 and 2 of the ITS sequences and the 5.8S rDNA sequence with primers ITS1 and and ITS4 [16] . Amplification reactions were performed in a 20 µl reaction mixture consisting of Solis BioDyne’s HOT FIREPol^® DNA Polymerase enzyme 4 µl, 1 µl of each primer and 13 µl of water.

The PCR program was adapted to that of the enzyme supplier as follows. An initial denaturation at 95˚C for 12 minutes, followed by 35 cycles of denaturation at 95˚C for 30 seconds, hybridization at 58˚C for 30 seconds and elongation at 72˚C for one minute, and final elongation at 72˚ for 5 minutes.

After amplification, the PCR products are revealed by electrophoresis on 10% agarose gel previously incorporated with Ethidium Bromide and illuminated with ultraviolet light (UV).

2.6. Sequencing

The PCR products of 111 DNA samples of fungal isolates revealed by agarose gel electrophoresis were sequenced by the biotechnology company Macrogen in the Netherlands.

2.7. Sequence Analysis

The sequencing results were first processed with BioEdit software for sequence alignment and cleaning. Using BLAST, the generated consensus sequence was then compared to other DNA sequences in the National Centre for Biotechnology Information (NCBI) non-redundant nucleotide database

3. Results3.1. Samples Collected

Sesame plant samples were collected from sesame production sites in 33 of the 45 provinces of Burkina Faso and across the three agroclimatic zones. Of a total of 149 diseased plant samples collected, 103 were from the Sudano-Sahelian zone, 28 were from the Sudanian zone and 18 were from the Sahelian zone.

3.2. Prevalence of Fungi Associated with Sesame in Burkina Faso

In general, depending on the organ of the diseased plant, the top five fungi encountered were as follows (Table 1):

- Roots: Macrophomina (96.40% of prevalence rate), Fusarium (22.30%), Phoma (9.35%), Cercospora (5.03%), Alternaria (4.32%);

- Leaves: Cercospora (79.16%), Macrophomina (56.25%), Fusarium (54.86%); Colletotrichum (46.52%), Phoma (41.66%);

- Stems: Macrophomina (87.83%), Fusarium (62.16%), Colletotrichum (45.27%), Cercospora (43.91%), Phoma (31.08%);

Table 1 Prevalence of fungal genera identified on samples and different plant organs of sesame collected in Burkina Faso

N˚	Fungi	Fungal prevalence rate (%)
N˚	Fungi	Wole Samples (N = 149)	Leave (N = 144)	Stems (N = 148)	Roots (N = 149)	Capsules (N = 126)
1	Alternaria	48.32	31.25	8.78	4.32	25.40
2	Botryodiplodia	26.85	8.33	13.51	4.32	9.52
3	Cercospora	86.58	79.17	43.92	5.04	42.86
4	Colletotrichum	61.07	46.53	45.27	4.32	7.94
5	Curvularia	31.54	25.00	9.46	1.44	19.05
6	Exserohilum	14.09	6.25	4.73	0.72	4.76
7	Fusarium	85.23	54.86	62.16	22.30	41.27
8	Macrophomina	97.99	56.25	87.84	96.40	65.87
9	Myrothecium	2.68	1.39	0.68	0.00	0.00
10	Nigrospora	26.85	19.44	2.04	0.72	9.52
11	Pestalotia guepini	5.37	3.47	0.00	1.44	1.59
12	Phoma	62.42	41.67	31.08	9.35	21.43
13	Phomopsis	6.04	0.70	3.38	0.72	2.38
14	Rhizoctonia solani	10.07	6.94	4.73	0.72	7.94
15	Inconnus	34.90	9.72	8.16	2.16	16.67
16	Cladosporium	20.13	-	-	-	-
	Aspergillus	-	-	-	-	-
17	Melanospora	2.68	-	-	-	-

- Capsules: Macrophomina (65.87%), Cercospora (42.85%), Fusarium (41.26%), Alternaria (25.39%), Phoma (21.43%).

In summary, these results show that Macrophomina, Fusarium, Cercospora, Colletotrichum, Phoma and Alternaria were the major fungi associated with diseased sesame plants in Burkina Faso.

Regarding the distribution of fungi among the climatic zones (Table 2), four (4) fungi, namely Cercospora, Colletotrichum, Macrophomina and Phoma, were widespread in all three climatic zones, contaminating between 50% and 100% of the samples collected in each zone. The fungi with the lowest occurrence in the zones were Myrothecium (0% - 3.88%), Phomopsis (0% - 10.71%) and Melanospora (0% - 11.11%). In general, all the fungi were invariably distributed among the three zones except Cercospora, Exserohilum and Cladosporium which were diversely distributed according to the climatic zones. Cercospora was more present in plants from the Sudano-Sahelian and Sudanian zones (91.26% - 92.86%) than in those from the Sahelian zone (55.56%). On the other hand, Exserohilum, and to a lesser extent, Cladosporium were more frequently found in samples collected in the Sahelian zone (38.89% each) than in those collected in the other two zones (9.71% - 14.29% and 14.56% - 28.57%, respectively).

Table 2 Prevalence of fungal genera identified on samples of diseased sesame plants according to the different agroclimatic zones

N˚	Genus of fungi	Probability (5%)	Prevalence of fungi by climatic zone (%)
N˚	Genus of fungi	Probability (5%)	Sudanese-Sahelian	Sudanian	Sahelian
1	Alternaria	0.4832	46.60a	60.71a	38.89a
2	Botryodiplodia	0.2014	24.27a	25.00a	50.00a
3	Cercospora	0.0001	91.26a	92.86a	55.56b
4	Colletotrichum	0.8713	62.14a	64.29a	50.00a
5	Curvularia	0.6066	32.04a	25.00a	38.89a
6	Exserohilum	0.0042	9.71b	14.29b	38.89a
7	Fusarium	0.2500	88.35a	85.71a	77.78a
8	Macrophomina	0.0977	99.03a	96.43a	100.00a
9	Myrothecium	0.4046	3.88a	0.00a	0.00a
10	Nigrospora	0.9698	26.21a	25.00a	33.33a
11	Pestalotia	0.2699	4.85a	10.71a	5.56a
12	Phoma	0.1450	58.25a	82.14a	61.11a
13	Phomopsis	0.3304	5.83a	10.71a	0.00a
14	Rhizoctonia	0.1718	7.77a	10.71a	22.22a
15	Cladosporium	0.0274	14.56b	28.57ab	38.89a
16	Melanospora	0.0528	1.94a	0.00a	11.11a
17	unknown	0.7207	35.92a	28.57a	38.89a

3.3. Molecular Identification of Fungi Associated with Diseased Sesame Plant Samples

Revealing PCR products by 10% agarose gel electrophoresis with a 100 base pair molecular weight marker yielded bands between 500 and 600 base pairs (Figure 2).

The results of the ITS sequence alignment followed by their comparison with the National Centre for Biotechnology Information non-redundant nucleotide database were presented in Table 3. The percentages of identity and coverage of the sequences were respectively between 97.13% and 100% and between 85% and 100%. As for the locus size of the corresponding closest accession, it ranged from 523 to 1120 base pairs. The results identified 25 species of fungi belonging to 13 genera which are Macrophomina, Fusarium, Cercospora, Corynespora, Alternaria, Colletotrichum, Nigrospora, Exerohilum, Lasiodiplodia, Curvularia, Phoma, Cladosporium and Didymela.

Sequence analysis revealed that all 20 isolates of the genus Macrophomina were close to those of the species Macrophomina phaseolina with percentages of identity and coverage between 95% and 100%, and between 97.47% and 100% respectively. In addition to these 20 isolates, one isolate identified molecularly as belonging to the genus Rhizoctonia was found to be very close to the reference

Table 3 DNA sequences analysis results by blast on NCI

Isolate	Morphological Identification	Isolate sequence size (bp)	Correspondant species in the NCBI database	coverage (%)	Similarity rate (%)	Accession size (BP)	Accession
MpTap-BF01	Macrophomina phaseolina	556	Macrophomina phaseolina	100%	99.82%	583	MT186826.1
MpGamp-BF02	Macrophomina phaseolina	548	Macrophomina phaseolina	100%	100.00%	583	MT186826.1
MpMpa-BF03	Macrophomina phaseolina	559	Macrophomina phaseolina	99%	100.00%	583	MT186826.1
MpSin-BF04	Macrophomina phaseolina	557	Macrophomina phaseolina	99%	100.00%	583	MT186826.1
MpTin-BF05	Macrophomina phaseolina	565	Macrophomina phaseolina	99%	98.45%	583	MZ502501.1
MpOuah-BF06	Macrophomina phaseolina	568	Macrophomina phaseolina	99%	99.65%	1120	OM106520.1
MpBou-BF07	Macrophomina phaseolina	558	Macrophomina phaseolina	99%	100.00%	583	MT186826.1
MpOuah-BF08	Macrophomina phaseolina	573	Macrophomina phaseolina	99%	99.48%	572	MW045603.1
MpGan-BF09	Macrophomina phaseolina	638	Macrophomina phaseolina	99%	97.47%	693	OM341626.1
MpLéo-BF10	Macrophomina phaseolina	556	Macrophomina phaseolina	99%	100.00%	583	MT186826.
MpCoal-BF11	Macrophomina phaseolina	553	Macrophomina phaseolina	100%	98.73%	596	MH864182.1
MpTib-BF12	Macrophomina phaseolina	554	Macrophomina phaseolina	100%	100.00%	693	OM341626.1
MpLéo-BF13	Macrophomina phaseolina	581	Macrophomina phaseolina	95%	97.49%	583	MT186826.1
MpNou-BF14	Macrophomina phaseolina	548	Macrophomina phaseolina	100%	98.72%	583	MT186826.1
MpHar-BF15	Macrophomina phaseolina	555	Macrophomina phaseolina	99%	99.10%	596	MH864182.1
MpSan-BF16	Macrophomina phaseolina	554	Macrophomina phaseolina	99%	100.00%	596	MH864182.1
MpKal-BF17	Macrophomina phaseolina	558	Macrophomina phaseolina	99%	99.28%	596	MH864182.1
MpSap-BF18	Macrophomina phaseolina	551	Macrophomina phaseolina	100%	100.00%	596	MH864182.1
MpPô-BF19	Macrophomina phaseolina	556	Macrophomina phaseolina	100%	99.82%	558	KF951702.1
MpTous-BF20	Macrophomina phaseolina	552	Macrophomina phaseolina	99%	99.82%	596	MH864182.1
MpGna-BF119	Rhizoctonia solani	555	Macrophomina phaseolina	99%	99.82%	596	MH864182.1
FusTin-BF21	Fusarium sp.	577	Fusarium incarnatum	92%	99.25%	543	MK192051.1
FusNiag-BF23	Fusarium sp.	569	Fusarium incarnatum	91%	99.61%	548	MT563420.1
FusMpa-BF22	Fusarium sp.	557	Fusarium fujikuroi	99%	99.46%	595	MT742817.1
Fus-Kamb-BF25	Fusarium sp.	599	Fusarium fujikuroi	98%	97.13%	592	MN565957.1
Fus-Mpa-BF24	Fusarium equiseti	617	Fusarium equiseti	85%	99.06%	551	MK764999.1
Fus-San-BF27	Fusarium equiseti	624	Fusarium equiseti	85%	99.07%	551	MK764999.1
Fus-Pô-BF29	Fusarium equiseti	709	Fusarium equiseti	100%	100.00%	571	MN498032.1
Fus-Kour-BF52	Fusarium equiseti	517	Fusarium equiseti	99%	100.00%	586	MT560375.1
FuSs-BF28	Fusarium sp.	617	Fusarium proliferatum	89%	99.27%	565	MT372093.1
Fus-Nia-BF36	Fusarium sp.	531	Fusarium proliferatum	99%	99.25%	572	OL873221.1
Fus-Bama-BF49	Fusarium sp.	523	Fusarium proliferatum	98%	99.61%	563	MT560218.1
Fus-Pô-BF33	Fusarium sp.	533	Fusarium proliferatum	99%	100.00%	572	OL873221.1
Fus-Nou-BF48	Fusarium sp.	527	Fusarium proliferatum	99%	99.62%	576	MT560212.1
Fus-Kom-BF38	Fusarium sp.	530	Fusarium penzigii	99%	99.62%	548	MN548457.1
Fus-Pam-BF40	Fusarium sp.	541	Fusarium penzigii	98%	99.44%	548	MN548457.1
Fus-Tap-BF47	Fusarium sp.	532	Fusarium penzigii	99%	98.68%	548	MN548457.1
Fus-Kour-BF30	Fusarium sp.	575	Fusarium oxysporum	98%	97.19%	687	KU671036.1
Fus-Sin-BF43	Fusarium sp.	708	Fusarium oxysporum	99%	99.62%	557	MN726603.1
Fus-Yam-BF41	Fusarium solani	537	Fusarium solani	100%	99.81%	553	MN545491.1
Cer-Mpa-BF59	Cercospora sp.	511	Cercospora sesami	99%	100.00%	545	MK764999.1
Cer-Pô-BF63	Cercospora sp.	514	Cercospora sesami	100%	98.81%	545	MN498032.1
Cer-Mpa-BF66	Cercospora sp.	1005	Cercospora sesami	100%	99.80%	545	MT560375.1
Cer-Pam-BF67	Cercospora sp.	1021	Cercospora sesami	99%	100.00%	545	MT372093.1
Cer-Kom-BF68	Cercospora sp.	512	Cercospora sesami	100%	100.00%	545	OL873221.1
Cer-Ded-BF70	Cercospora sp.	545	Cercospora sesami	99%	100.00%	545	MT560218.1
Cer-Tin-BF71	Cercospora sp.	707	Cercospora sesami	99%	100.00%	545	OL873221.1
Cer-Komb-BF122	Cercospora sp.	507	Cercospora sesami	100%	99.80%	545	MT560212.1
Cer-Tib-BF54	Cercospora sp.	509	Cercospora kikuchii	100%	100.00%	550	MN548457.1
Cer-Kom-BF55	Cercospora sp.	1000	Cercospora kikuchii	99%	100.00%	544	MN548457.1
Cer-Sak-BF56	Cercospora sp.	876	Cercospora kikuchii	99%	100.00%	550	MN548457.1
Cer-Kom-BF57	Cercospora sp.	505	Cercospora kikuchii	99%	99.80%	545	KU671036.1
Cer-Léo-BF58	Cercospora sp.	511	Cercospora kikuchii	99%	99.80%	550	MN726603.1
Cer-Bama-BF72	Cercospora sp.	637	Cercospora canescens	99%	99.61%	545	MN545491.1
Cor-Gan-BF53	Cercospora sp.	538	Corynespora cassiicola	99%	99.81%	547	MW165772.1
Cor-Nia-BF61	Cercospora sp.	527	Corynespora cassiicola	100%	99.24%	570	MH762895.1
Cor-Komb-BF64	Cercospora sp.	1136	Corynespora cassiicola	99%	99.43%	561	MN945374.1
Cor-Bama-BF65	Cercospora sp.	756	Corynespora cassiicola	99%	99.81%	561	MN945374.1
Cor-Pô-BF69	Cercospora sp.	1130	Corynespora cassiicola	99%	100.00%	570	MH762895.1
Cor-Pô-BF63	Cercospora sp.	514	Corynespora cassiicola	99%	100.00%	561	MN945374.1
Cor-Léo-BF108	.Inconnu	529	Corynespora cassiicola	100%	99.81%	561	MN945374.1
Alt-Kamb-BF73	Alternaria sp.	663	Alternaria porri	99%	99.63%	582	MT554514.1
Alt-Léo-BF76	Alternaria sp.	666	Alternaria porri	99%	99.63%	582	MT554514.1
Alt-Boul-BF80	Alternaria sp.	542	Alternaria porri	99%	100.00%	582	MT554514.1
Alt-Ded-BF82	Alternaria sp.	542	Alternaria porri	99%	100.00%	582	MT554514.1
Alt-Pô-BF77	Alternaria sp.	553	Alternaria simsimi	98%	99.09%	571	JF780938.1
Alt-Sou-BF78	Alternaria sp.	551	Alternaria simsimi	99%	99.27%	571	JF780938.1
Alt-Bama-BF79	Alternaria sp.	559	Alternaria simsimi	98%	99.09%	571	JF780938.1
Alt-Komb-BF81	Alternaria sesami	554	Alternaria simsimi	98%	99.82%	571	JF780938.1
Alt-Nia-BF83	Alternaria sesami	549	Alternaria simsimi	99%	99.63%	571	JF780938.1
Alt-Pô-BF62	Alternaria sesami	544	Alternaria simsimi	100%	99.08%	571	JF780938.1
Col-Tin-BF88	Colletotrichum gloeosporioides	544	Colletotrichum gloeosporioides	100%	99.63%	583	MW603454.1
Col-Gan-BF90	Colletotrichum capsici	572	Colletotrichum truncatum	98%	98.58%	586	KX685450.1
Col-Nou-BF91	Colletotrichum capsici	554	Colletotrichum capsici	99%	93.87%	592	MT012102.1
Cur-Léo-BF95	Curvularia lunata	582	Curvularia lunata	99%	99.48%	576	KU715116.1
Cur-Ded-BF96	Curvularia lunata	544	Curvularia lunata	99%	100.00%	562	LC317566.1
Cur-Bou-BF97	Curvularia lunata	594	Curvularia lunata	100%	98.66%	603	MH010914.1
Cur-Léo-BF98	Curvularia sp.	544	Curvularia lunata	100%	99.45%	573	MN173127.1
Pho-Sin-BF99	Phoma lingam	518	Phoma multirostrata	99%	100.00%	523	MT635199.1
Pho-Léo-BF100	Phoma lingam	531	Phoma multirostrata	99%	99.06%	547	KJ767077.1
Pho-Sap-BF102	Phoma lingam	518	Phoma multirostrata	99%	100.00%	547	KJ767077.1
Pho-Pô-BF103	Phoma sorghina	520	Phoma multirostrata	99%	99.81%	524	KM659039.1
Pho-Nia-BF104	Phoma sorghina	526	Phoma multirostrata	99%	99.23%	524	KM659039.1
Nig-Gan-BF112	Nigrospora oryzae	529	Nigrospora sphaerica	99%	92.67%	559	MW081353.1
Nig-Har-BF114	Nigrospora oryzae	534	Nigrospora oryzae	99%	95.68%	532	MT672515.1
Exs-Sin-BF105	Exserohilum rostratum	580	Exserohilum rostratum	100%	100.00%	603	MN960317.1
Exs-Kom-BF118	Exserohilum rostratum	580	Exserohilum rostratum	100%	99.59%	627	MN599590.1
Las-Sap-BF116	Botryodyplodia theobromae	518	Lasiodiplodia theobromae	99%	99.81%	550	MK530050.1
Cla-Tib-BF120	Cladosporium sp.	524	Cladosporium sphaerospermum	99%	99.81%	530	MF467891.1
Did-Kak-BF74	Inconnu	6542	Didymella americana	99%	99.24%	550	MK646045.1

Mp = Macrophomina phaseolina; Fus = Fusarium; Cer = Cercospora; Cor = Corynespora; Alt = Alternaria; Col = Colletotrichum; Cur = Curvularia; Pho = Phoma; Nig = Nigrospora; Exs = Exserohilum; Las = Lasidiodiplodia; Cla = Cladosporium; Did = Didymella; BF = Burkina Faso; 2nd three correspond to the collecting site name.

isolate of M. phaseolina (MH864182.1) with a percentage identity of 99.82%. Eight (8) of the Macrophomina isolates showed perfect similarity (100% identity) with the M. phaseolina isolate MH864182.1 in the database.

Out of the 30 isolates of the genus Fusarium, the analysis of the sequences obtained identified 19 isolats belonging to seven (7) species including F. proliferatum (5), F. equiseti (4), F. penzigii (3), F. incarnatum (2), F. fujikuroi (2), F. oxyporum (1) and F. solani (1). Sequence identity and coverage percentages of Fusarium isolates ranged from 97.18% to 100% and 85% to 100%, respectively. Two Fusarium isolates showed complete similarity to the reference isolate (MN498032.1) of F. equiseti, and one to the reference isolate (OL873221.1) of F. proliferatum.

Three (3) species of Cercospora including C. sesami (8 isolates), C. kikuchii (5) and C. canescens (1) were identified after sequence analysis of the isolates belonging to this genus, with percentages of identity and coverage of 99.61% - 100% and 99% - 100%, respectively. The eight (8) isolate sequences identified as those of C. sesami species were close to that of a single accession (MT186826.1) in the NCBI database. Of these eight isolates, five showed perfect similarity to the reference accession of C. sesami. Two isolates were also identical to the reference isolate MK336506.1 of C. kikuchii, and one was identical to the reference isolate MH777047.1 of C. kikuchii. Sequence analysis also revealed a new genus: Corynespora grouping the six other isolates initially identified morphologically as belonging to the genus Cercospora. These isolates all belong to the species Corynespora cassiicola, two of them being perfectly similar to the NCBI reference isolates MH762895.1 and MNP45374.1 of C. cassiicola. One isolate belonging to the genus “Unknown” was also identified as C. cassiicola.

All DNA sequences from Alternaria isolates were identified as closely related to A. simsimi and A. porri. A comparison of the sequences to the NCBI nucleotide database indicated that four were very close to the sequence of the accession (MT554514.1) of A. porri with a similarity rate of 100% for two of them. As for the other sequences, six (6), belonging to A. simsimi, the percentages of coverage were between 98% and 100% and the percentages of identity between 99.08% and 99.82%. These sequences were also close to accession JF780938.1 of the database.

Colletotrichum gloesporioides, Colletotrichum truncatum and Colletotrichum capsici were the three species identified after sequence analysis of Colletotrichum isolates, with coverage and identity percentages of 100% and 99.63%, 98% and 98.85%, and 99% and 93.87%, respectively with their respective reference sequences MW603454.1, KX685450.1 and MT012102.1. Molecular analysis of the remaining seven isolates of the Colletotrichum genus was inconclusive.

With coverage percentages of 99% and identity rates between 99% and 100%, the DNA sequences of the five isolates of the genus Phoma were very close to those of two reference accessions (MT635199.1, KJ767077.1) of the species Phoma multirostrata, with perfect similarity for two of the isolates.

Of the five isolates belonging to the genus Curvularia, four were identified as closely related to the species C. lunata, with varying percentages of identity (93.87% - 100%). Of these isolates, only one showed 100% identity with the reference accession LC317566.1 in the database. Analysis of the fifth isolates was inconclusive.

The two Nigrospora isolates were all identified as Nigrospora sphaeriaca (MW081353.1) and Nigrospora oryzae (MT672515.1), with reference species identity percentages of 92.67% and 95.68%.

The two isolates of the genus Exserohilum were all identified as closely related to two accessions (MN960317.1 and MK530050.1) of the species Exserohilum rostratum, with, however, one of the isolates identical to accession MN960317.1 in the database.

Three isolates initially identified morphologically as belonging to the genera Botryodiplodia, Cladosporium, and “unknown” were molecularly identified as Lasiodiplodia theobromae, Cladosporium sphaerospermum and Didymella americana, respectively. The sequences showed near 100% coverage and 99% similarity rates to accessions of the three respective reference species.

Molecular analysis of isolates initially identified as belonging to the genera Pestalotia (1 isolate), Phomopsis (1); Melanospora (1) and “unknown” (7), yielded inconclusive results.

4. Discussion

One of the advantages of sesame is that it can be produced under a variety of climatic conditions ranging from dry arid zones to humid and rainy zones [17] . Sesame is produced throughout Burkina Faso, across the country’s three agro-climatic zones. However, the largest sesame-producing provinces are located in the Sudano-Sahelian zone, making this area the most important for the production of this important cash crop for the country. Thus, the collection of samples of diseased sesame plants focused on this zone with 102 collection sites without forgetting the other zones in proportion to the importance of their production.

Based on morphological characteristics, several species of fungi belonging to 16 genera were identified as associated with samples of diseased sesame plants, reflecting the diversity of potential pathogenic fungi associated with sesame in Burkina Faso. In Pakistan, the diversity of pathogenic fungi in sesame had been reported by Altaf et al. [18] who identified 11 species of pathogenic fungi associated with poor seed germination and diseased sesame seedlings.

The diseased plant samples were heavily contaminated by the genera Macrophomina (97.99%), Fusarium (85.23%), Cercospora (86.58%), Phoma (62.42%) and Colletotrichum (61.07%) with prevalence rates above 50%. These high prevalence rates reveal the importance of the species of fungi of these genera in sesame production sites in Burkina Faso. When considering the different plant organs of the samples, the predominance of these genera was variable. On roots, stems and capsules, Macrophomina was the dominant genus, while Cercospora was the most important on leaves. Species belonging to both genera have been reported as major pathogens on sesame. The genus Macrophomina and particularly the species M. phaseolina is a pathogen, responsible for root and stem rot (known as ash rot) on several crops of economic importance [19] [20] including sesame [10] [11] . One of the important leaf diseases of sesame is due to the species Cercospora sesami responsible for the so-called Cercospora Leaf Spot (CLF) [12] [21] .

In addition to the genera Macrophomina, Fusarium and Colletotrichum, all sesame stems pathogens [14] , Cercospora has also been strongly detected on sesame stems from Burkina Faso. The importance of the Cercospora genus on stems suggests a high severity of the disease, greater than or equal to 43.1% according to the rating scale of Enikuomehin et al. [12] corresponding to the appearance of symptoms on the stem.

According to the different agroclimatic zones of sample collection, the study revealed the strong presence of five genera (Cercospora, Colletotrichum, Fusarium, Macrophomina and Phoma) with incidence rates ≥ 50% in all zones. These fungi are known as causal pathogens of the destructive sesame diseases Cercospora Leaf Spot (Cercospora), anthracnose (Colletotrichum), fusarium wilt disease [22] root and stem rot (Macrophomina), leaf spot (Phoma) [14] . The expansion of these fungi in all areas suggests that these microorganisms adapt to a wide range of moisture and temperature conditions. It is noteworthy that all samples from the Sahelian zone were contaminated with the genus Macrophomina. The species M. phaseolina has been recognized as responsible for ash rot on sesame by several authors [10] [11] . The development of this disease would be favoured by high temperatures [23] [24] and pockets of drought, characteristic of this part of the country. Exserohilum and Cladosporium are genera that are particularly represented in the Sahelian zone characterized by annual rainfall of less than 600 mm and high temperatures, indicating that dry and hot conditions seem favorable to the development of these two genera. In general, all other genera invariably proliferate in both the Sudanian and Sudano-Sahelian zones.

Molecular tools used for the identification of fungi, due to their accuracy, are often complementary to identification based on morphological characteristics. Identification of fungi isolated from fragments of diseased sesame plants based on morphological characteristics could easily lead to misidentification. Based on the morphological characteristics of the 111 isolates obtained, were identified as belonging to 16 known genera including Fusarium, Macrophomina, Cercospora, Alternaria, Colletotrichum, Phoma, Curvularia, Nigrospora, Cladosporium, Exserohilum, Pestalotia, Phomopsis, Rhizoctonia, Melanospora, Myrothecium, and Botryodiplodia, and other unidentified genera referred as “Unknown” genus. DNA sequence analysis of these same fungal isolates identified 25 species belonging to 11 of the 16 genera initially identified morphologically, thus confirming the diversity of the mycoflora associated with sesame and revealed by the morphological identification. Three new genera were also identified. These are the genera Corynespora, Lasiodiplodia, and Didymella.

The 20 isolates of the genus Macrophomina were identified as Macrophomina phaseolina. Molecular analysis also showed that the isolate previously identified morphologically as belonging to the genus Rhizoctonia was found to be closely related to the species M. phaseolina. These results indicate that M. phaseolina species is probably the only species of the genus Macrophomina associated with sesame in Burkina Faso. The uniqueness of species in this genus could be explained by the use of generic primers and not specific to this genus. The work of [25] developed primers specific to three species of the genus Macrophomina including M. phaseolina.

From the 30 isolates of the genus Fusarium, molecular analysis confirmed 19 as belonging to the genus Fusarium and composed of seven different species, thus revealing a diversity of Fusarium species associated with diseased sesame plants in Burkina Faso. These are F. proliferatum (5 isolates), F. equiseti (4), F. penzigii (3), F. incarnatum (2), F. fujikuroi (2), F. oxysporum (2) and F. solani (1). All species identified, except F. penzigii have been previously reported as pathogens of sesame [14] . These include F. proliferatum [27] , F. oxysporum [13] [22] [27] , F. solani [28] , F. equiseti and F. fujikuroi [14] . Among these species, F. oxysporum responsible for fusarium rot of sesame is one of the major pathogenic fungi in the production areas of major sesame-producing countries [22] [29] . It should be noted that molecular analysis of the other 11 isolates, initially identified as belonging to the genus Fusarium, was inconclusive.

Molecular analysis revealed three species of Cercospora: C. sesami (8), C. kikuchii (5) and C. canescens (1). The remaining six Cercospora isolates and one unknown isolate were found to be Corynespora cassiicola. One of the most prevalent diseases of sesame in the production areas is CLF due to Cercospora sesami [12] [31] [32] . In addition to this species, C. kikuchii and C. canescens were identified on diseased plant samples, suggesting the presence of three probable species associated with sesame leaf necrosis in Burkina Faso.

Alternaria Leaf Spot (ALS) due to Alternaria sesami and Alternaria sesaminicola is the major leaf disease of sesame in the humid tropics. In the present study, two potential species of ALS agents of sesame were identified. These are A. simsimi previously reported as the cause of ALS in Korea [32] and A. porri reported by [33] as the cause of ALS of onion.

Apart from the genera Fusarium, Cercospora and Alternaria reported as the major fungal pathogens of sesame worldwide, the present study identified species of the genera Colletotrichum (2), Curvularia (1), Phoma (1), Nigrospora (2), Exserohilum (1) associated with sesame from Burkina Faso and previously reported by Enikuomehin et al. [14] as potential pathogens of sesame. Accurate molecular identification allowed the identification of the species Corynespora cassiicola reported to cause spots on leaves, stems, roots and flowers of several economically important plants [34] . On the sesame crop, [35] reported for the first time in China, the root rots due to C. cassiicola. In the present study, C. cassiicola was associated with all parts of the plant but particularly leaves and would be a potential foliar disease agent on sesame [36] .

Based on morphological and molecular characteristics many potential pathogenic fungi belonging to many genera are identified associated with the sesame plant in Burkina Faso.

5. Conclusion

Morphological identification of fungi associated with samples of diseased sesame plants demonstrated a diversity of potential pathogen agents of sesame in Burkina Faso. This diversity varies according to the agro-climatic zones of the country and is composed of 16 genera dominated by Macrophomina, Fusarium and Cercospora. Molecular identification confirmed most of the results obtained from the morphological identification, providing precision on the identity of the fungal species associated with sesame in Burkina Faso. Thus, the top three fungal genera associated with sesame in Burkina Faso are Macrophomina, Fusarium and Cercospora. A study of the pathogenicity of the main species identified and further investigations on the genetic diversity of the isolates by using specific primers are necessary for the development of effective protection methods against the main diseases of the crop.

Acknowledgements

The authors express their gratitude to the Plant Pathology Laboratory of the Center for Environmental and Agricultural Research, and Training (CREAF) in Kamboinsé for the financial support in the diseased plant samples collection. They thank the Laboratoire Mixte Internationale (LMI) Patho-Bios for the use of its molecular biology platform and financial support. They also express their gratitude to Ezéchiel Bionimian TIBIRI, Séni BILGO and Marcelin TIEMTORE for their technical support.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

Cite this paper

Soalla, W.R., Zida, P.E., Neya, B.J. and Koita, K. (2023) Morphological and Molecular Identification of Fungi Associated with Sesame Diseased Plants of the Three Agroclimatic Zones of Burkina Faso. American Journal of Plant Sciences, 14, 290-307. https://doi.org/10.4236/ajps.2023.143020

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