Introduction

Ivorian sugar production amounts to 330,000 t. The cultivation of sugar cane is subject to several constraints. The pathologies associated with this crop are caused by viruses, stem-boring insects, bacteria and fungi¹. The monoculture practiced for several years in the sugar complexes leads to a decline in soil fertility. All of these factors contribute to a drop in production². Biotic constraints have caused reductions in yield in the sugar industries of Côte d’Ivoire, notably those of Borotou-Koro and Zuénoula. Fight against these pathogens, pesticides, especially synthetic fungicides such as benzimidazoles, triazoles and strobilurins, are mainly used. However, the excessive use of these products in sugar cane plantations could encourage the appearance and proliferation of resistant strains, leading to an increase in diseases³. Faced with these harmful effects, it is essential to precisely identify and know the pathogenic fungal profile, in order to effectively fight against these phytopathogens responsible for serious pathologies. The fungal agents responsible for these diseases are often unknown. It is within this framework that this study falls, the general objective of which is to establish the profile of pathogenic fungal strains of sugar cane in Côte d’Ivoire.

Materials and methods

Material

Organs (stems, leaves and shoots) of sugar cane presenting pathologies were collected in the village plantations of Borotou-koro (08°31 North latitude and 7°17 West longitude) and Zuénoula (7°30 and 7°40 North latitude, and between 6°50 and 6°15 West longitude) in Côte d’Ivoire.

Sampling

Samples of shoots, leaves and stems showing symptoms of pathology were collected. Thus, 5 samples consisting of 5 leaves, 5 stems and 5 shoots presenting pathologies were collected per plantation. Or 50 samples at a rate of 25 samples per production area. The collected samples were packaged in sterile stomacher bags, labeled, sealed then kept in a cooler containing cold accumulators where the temperature was from 4°C.

Phenotypic identification of pathogens

Isolation was carried out according to the method of Silué et al⁴. Thus, the sugarcane organ fragments were sterilized in ethanol (70%) during one minute, immersed in 3% bleach for 3 minutes, and rinsed with sterile distilled water three times. The fragments collected were inoculated by direct contact on medium Potato Dextrose Agar with Chloramphenicol. The culture were then incubated at 27±1°C for 24 to 72 hours. Macroscopic identification was carried out according to the method of  Botton et al⁵ by examining the culture. The cultural characteristics determined were the texture, the color of the thallus and the color of the underside of the crop. As for the microscopic identification, it was carried out according to the method described by Guiraud⁶ taking a filament using sterile tweezers then placed in a drop of methylene blue, placed on object slide and covered with a coverslip then observed under a LEICA DM750 optical microscope with an X40 objective. The characteristics observed were the appearance of the mycelium (compartmentalized or not), the presence and shape of the spores (oval, spherical, round, etc.), the shape of the conidial heads and the size of the conidiospores (short or long).

Molecular identification of pathogens

DNA extraction

Fungi colonies were collected and bubbled in 1 ml of sterile water. The suspension thus obtained is centrifuged at 12,000 rpm for one minute. The pellet of this suspension was collected and suspended in 200 μl of Chelex 100 (Bio-Rad) previously prepared following to the manufacturer’s instructions. The new suspension obtained was homogenized using a vortex for ten seconds. Heating in a water bath at 100°C for eight minutes was carried out followed by sudden cooling in ice. This thermal shock allows the lysis of cells and the release of DNA. A new homogenization step using a vortex for ten seconds was carried out at 12,000 rpm for two minutes. In the base are the proteins captured by the Chelex 100 and in the supernatant is the DNA. DNA has been preserved at -20°C for PCR amplification.

Polymerase Chain Reaction

Primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’ TCCTCCGCTTATTGATATGC-3’) were used. For the preparation of the reaction mixture, an Invitrogen kit was used. This kit consists of Taq DNA polymerase, polymerase buffer and MgCl2⁷. dNTPs and primers were also used. Final volume of  amplification reaction was 50 μl. PCR  medium was composed of 26.35µL of MilliQ Water, 5µL of  ITS1, 2.5 μL of ITS4, 0.25 μL of Taq polymerase (Promega) and 10 μL of template DNA. The amplification reaction was made in a thermal cycler (Techne® Prime Thermal Cycler Range, USA) according to the program described by Bessadat et al⁸. The first step is that of initial denaturation. It is done at 94°C for five minutes. The second stage contains 35 cycles, each of which is composed of a denaturation phase at 94°C for thirty seconds, annealing phase at 55°C for thirty seconds and elongation phase at 72°C. Finally, the third stage is that of the final elongation which is done at 72°C for ten minutes.

Profile visualisation

The PCR profile were revealed by electrophoresis on 1.5% agarose gel. For gel preparation, the agarose was suspended in 50 ml of 0.5X TBE. The suspension was heated and left to room temperature for five minutes then a drop of ethidium bromide was incorporated before pouring it into a gel holder. Once solidified, the gel was immersed in an electrophoresis tank containing 0.5X TBE. 10 μl of amplified fragments were mixed with 2 μl of loading buffer (50% TE, pH 8; 50% glycerol; bromophenol blue) before being loaded into the gel wells. Profile was visualized using a transilluminator (Vilber Lourmat) under UV illumination after migration at 90 Volts for 90 minutes. The size of the DNA fragments was determined using the Reddy Run Super ladderlow 100 bp molecular size marker (Thermo Scientific) used as a reference.

Sequencing

Sequencing was carried out from the rDNA products amplified with an automated sequencer. The primers used for the amplification of the regions studied were also used as sequencing primers. All DNA sequences obtained during sequencing were combined in a database (GenBank), used for the reconstruction of taxonomic relationships between the species studied. The homology of related sequences was investigated using the bioinformatics search tool BLAST. Multiple sequence alignment was carried out using Clustral W software⁹.

Pathogenicity of isolated fungi

Preparation of fungal inoculum

Ten milliliters (10 mL) of sterilized distilled water were added to 14-day-old mycelial cultures of pathogenic fungi. The surface of these cultures was scraped using a sterilized metal spatula. The resulting solution was homogenized in a test tube using a vortex. A few drops (200 µL) of this suspension were used to fill the wells of a Malassez cell in order to estimate the number of spores. This number was adjusted to 10⁶ spores/ml by dilution or by increasing the spore concentration of the solution by returning the spore solution to another mycelial culture. One milliliter (1 mL) of a 1% glucose and agar solution is added to the spore suspension before inoculation. The role of this solution is to facilitate the adhesion and germination of spores on the leaves¹⁰.

Infection of sugarcane seedlings with spore suspensions of pathogenic fungi

Using a syringe, the underside of the leaves of sugarcane seedlings was infected with 1 mL of inoculum of the pathogenic fungus. The greenhouse was regularly humidified (twice a day) by watering using a watering can in order to maintain the high humidity level (95 – 100%). The purpose of humidification was to facilitate the germination of spores of the fungal pathogen. The evolution of fungal disease symptoms was monitored regularly for three (3) months. Symptomatic organ samples were collected for the isolation of inoculated pathogenic fungi¹⁰.

Results

Microscopic pathogenic fungi of sugar cane

Sugar cane samples are attacked by molds presenting colonies of various appearances, textures and colors. Thus, 129 fungal isolates were isolated taking into account the resemblance of thalli and spores. Phenotypic identification according to the macroscopic characteristics of the colonies and on the basis of the microscopic characteristics of the mycelium and conidia or spores made it possible to highlight 2 genera, namely Fusarium and Schizophyllum (Table 1).

Table 1: Phenotypic characteristics of isolated genera.

Molecular characteristics of sugarcane pathogenic fungal isolates

Sequence analysis made it possible to identify 3 species with similarity rates of 99 % (Table 2).  Thus, we obtained Schizophyllum commune (I), Fusarium verticillioides (II) and Fusarium subglutinans (III).

Table 2: Fungi strains identified from sugar cane samples

Nc: number of nucleotides compared. %H: percentage of sequence homology

Pathogenicity of fungal isolates

Pathogenicity test is presented in Table 3. All species were found to be pathogenic to sugarcane. Thus, Fusarium verticillioides presented the symptoms of red movre disease characterized by reddish spots and Fusarium subglutinans induced the characteristic symptoms of Pokkah Boeng disease. Schizophyllum commune caused black rots characteristic of anthrax. (Table 3).

Table 3: Pathogenicity test Click here to view Table

Discussions

Identification of pathogenic fungal strains was carried out based on phenotypic and molecular characteristics. During this work, several species of Fusarium were identified. Their presence explains their involvement in the “Pokkah Boeng” disease. Indeed, the work of Hilton et al¹¹ and those of Lin et al¹² showed that several Fusarium species have been reported as causative agents of “Pokkah Boeng”, including F. sacchari, F. fujikuroi, F. verticillioides, F. andiyazi, F. proliferatum and F. moniliforme. The identification of Fusarium verticillioides on canes in the locality of Zuénoula would implicate it in the transmission of red snot. The samples collected in this locality showed red spots. Thus, Suresh and Nelson¹³ showed that the causative agent of red snot was Fusarium verticillioides. These results agree more or less with those of Kouadia et al¹⁴ who highlighted a diversity of phytopathogenic fungi belonging to the genera Colletotrichum, Fusarium, Aspergillus, Phoma, Penicillium, Curvularia, Botryodiplodia and Rhizoctonia associated with spoilage of banana, avocado and mango fruits. Furthermore, the genera Fusarium, Aspergillus, Botryodiplodia and Phoma have been reported on avocado and mango fruits¹⁵. These fungi are known to cause the deterioration of many products such as fruits, seeds, vegetables and tubers both in the field and during storage¹⁶. The fungal diversity recorded could be explained by the fact that sugar cane is a food with high nutritional value, mainly consisting of carbohydrates, water, vitamins and minerals.  Also, the pH of sugar cane also being lower than 4.6, would more favor the development of pathogenic fungi¹⁷.  Naturally, sugar cane resists microbial attacks, the extent of which will depend on the stage of maturity. This is because sugar cane contains high concentrations of natural antimicrobial compounds. These compounds consist of mixtures of 5-substituted resorcinols such as resorcinol-5-(12-heptadecadienyl) and resorcinol-5-(pentadecyl) Prusky et al¹⁸ accumulate in the stem. Faced with such a chemically unfavorable environment, the fungus suspends its development until the concentration of antimicrobial compounds decreases in the stem. The results of pathogenicity tests carried out on the canes showed that all of these isolated fungi were pathogens of sugarcane. Schizophyllum commune was the species that caused the most serious pathologies of sugar cane in the two localities after the Mosaic. This could be explained by the fact that this species is involved in anthrax. These results are in agreement with those of Yao et al¹⁹ who showed that the pathology spread very quickly in all sugar cane producing countries causing numerous yield losses ranging from 30 to 50% for susceptible varieties.

Conclusion

This present study contributed to the identification of pathogenic fungal germs of sugar cane in Côte d’Ivoire. From this study, it should be noted that the fungal species, namely Fusarium verticillioides, Fusarium subglutinans and Schizophyllum commune, were identified as pathogens of sugar cane in Côte d’Ivoire. These fungi are believed to be involved in pathologies such as red snot, “pokkah boeng” and smut, which are commonly encountered in sugar cane plantations.

Acknowledgment

The authors are thankful for the sugar society SUCRIVOIRE of Côte d’Ivoire.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Permission to reproduce material from other sources

Not Applicable 

Author Contributions

Djédji Okoue, Armist Amoa Tehua and Ibrahima Kebe : Performed the majority of the experiments and data analysis.

Mireille Waze Alloue-Boraud : Designed the experiments and analysed the data.

Fulgence Yao Koffi : Wrote the manuscript.

All authors read and approved the final version of the manuscript.

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