Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane

INTRODUCTION

Sugarcane is one of the top ten food crops in the world and an important economic crop in many tropical countries [1]. Sugarcane is infested with many plant pathogens including fungi, bacteria, and viruses as well as, insect pests. The genus Fusarium is one of the most devastating plant pathogens producing several mycotoxins [2]. Two species of Fusarium including F. subglutinans and F. sacchariare well reported to cause Pokkahboeng disease of sugarcane. Fusarium species are able to produce numerous phytotoxins and mycotoxins that could be causing major diseases in humans, animals, and plants [3-5]. Mycotoxins were produced by Fusarium species and other fungi comprising moniliformin, fusaproliferin, fusarins, butenolides, beauvericin, enniatins, and fusaric acid, etc [6-10]. One of interesting mycotoxins is a beauvericin affecting for health of animals and human due to this toxin is more common contaminant for grains after infection with Fusarium [10-11].

Beauvericin (BEA) is a bioactive cyclodepsipeptide that contains three of N-methyl-L-phenylalanyl and D-a-hydroxy-isovaleryl [12]. It was first isolated from entomopathogenic fungi Beauveriabassiana[13-14]. However, the first detected BEA of Fusarium species was by Gupta and coauthors [15]. It is limiting data on the occurrence and toxicity of BEA compared with other mycotoxins. BEA was considered as insecticidal and phytotoxic that exhibited several biological properties [15], antibacterial [16], as well as, showing cytotoxicity towards cell lines from invertebrates, animals and human [17]. The methods for the detection of mycotoxins include gas chromatography (GC), GC-MS, enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF/TOF), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and tinlayer chromatography (TLC) [18-21]. TLC is using a qualitative screening method that assayed many mycotoxins [22].

Several methods have been used for determining the toxicity of mycotoxins by bioassay test. Bioassay could test on organisms including microorganisms, insects, aquatic animals, insects, plants, tissue culture, and organ. There are three bioassays systems including brine shrimp, zebrafish, and chicken embryo known to be able to detect more than 10 types of mycotoxins [23]. The popular choice for the bioassay test is brine shrimp according to the high sensitivity to mycotoxin [24-25]. Therefore, the study aims to identify the Fusarium isolates and investigate their ability for producing BEA. Then, the biotoxicity of BEA was determined using brine shrimp (Artemiasalina).

MATERIALS AND METHODS

Culture of Fusarium isolates from sugarcane A total of 55 isolates for Fusarium spp. were reculturedfrom the Fusarium culture collection unit, School of Biology Science, University Sains Malaysia, Penang, Malaysia. These isolates were taxonomically identified in a previous study [26] by using the Fusarium laboratory manual for Leslie and Summerell [2].

Identification of the F. subglutinans and F. saccharifrom sugarcane

Morphological characteristics were used to identify F. subglutinans and F. sacchari isolates to reconfirm their identity from the stock culture. The morphological descriptions were based on Booth [27], Gerlach and Nirenberg [28], Nelson and coauthors [29], Burgess and coauthors [30], Leslie and Summerell [2], and Wijayawardeneand coauthors [31]. To study microscopic characteristics, the isolates cultured onto CLA (Carnation Leaf Agar) for 7 to 10 days [32]. Morphological features observed for identification of Fusarium species:

A. Macroconidia: Presence or absence, overall shape, shapes of apical and basal cell and the number of septa.

B. Microconidia: Presence or absence, shape, and the number of septa.

C. Mesoconidia: Presence or absence.

D. Conidiophores: Presence or absence, monophialides, and polyphialides. E. Chlamydospores: Presence or absence, mode of formation, and cell wall.

All the characteristics were observed using a light microscope (Olympus model BX-50F4) and photographed using a camera (JVC model KY-F55BE) with an image Analyzer-single image stereogram (SIS) program. For macroscopic characteristics, PDA (Potato Dextrose Agar) was used for observation of culture appearances, such as the texture of the colony, colony colour, pigmentations, and growth pattern. Mycelial disc of 6 mm diameter was plated onto the fresh PDA plate and the growth rates were recorded after 3 days of incubation. The culture appearance of each isolate on PDA was visually assessed after the mycelia were fully grown. The determination of colony colour and pigmentation was based on the colour description in the Methuen handbook of colour chart [33].

Cultivation of fungal isolates for mycotoxin screening

The Fusarium isolates were transferred to PDA plates and incubated for 7 to 10 days. Corn grits were used as a culture medium to analyses the presence of mycotoxin as it enhances the mycotoxin production. 15 g of corn grits with 45% of moisture was autoclaved in 250 ml Erlenmeyer flask. Spore suspension (approx. 1x10⁶ spores) of 1-2 week old culture was prepared to inoculate on the autoclaved corn grit. The inoculated corn grit and control (uninoculated corn girt) were incubated in dark at room temperature for 28 days. Control was treated the same except inoculants was substituted with substituted with sterilized double distilled water.

Extraction of Beauvericin (BEA)

BEA was extracted from 28 days old inoculated corn grit following the procedure of Logriecoand coauthors [34]. 15 g of corn grits were extracted overnight with 75 ml of acetonitrile, methanol, and water (16:3:1) and grounded in a warring blender for 5 min. The crude extracted was filtered through Whatman no.4 filter paper that was defatted twice with 25 ml of n-heptane. The bottom layer was evaporated to near dryness at 80°C by a rotary evaporator (Buchi 461, Switzerland). The residue was dissolved in 50 ml of a mixture of methanol and water with 1:1 proportion. Extraction was carried out twice using 25 ml of dichloromethane, which was evaporated and re-dissolved in 50 ml mixture of methanol and water with 1:1 proportion. Extraction was carried out twice using 25 ml of dichloromethane. BEA in the dichloromethane was evaporated and re-dissolved in 1 ml of methanol prior to the detection of BEA through TLC. The extracted mycotoxins were stored in a refrigerator.

Detection of Beauvericin (BEA)

The detection of BEA was carried out by using thin layer chromatography (TLC). TLC is a sensitive method for mixture analysis by separating the compound in the mixture. About 5 to 10 µl of extract were spotted onto a 20 x 20 cm silica TLC plates (Pre-coated with silica gel 60 F254; E. Merck AG, Darmstadt, Germany) along with the standard of BEA at 200 ppm from Sigma, USA. Plates developed in the solvent system according to Song and coauthors [35] in a mixture of acetic acid, methanol, water (100:5:1). The plate was air-dried and subsequently, the spot on the TLC plate detected by iodine vapour. Retention factor (Rf) values for the standard and samples were measured. The Rf values were calculated according to Touchstone [36] and Fessenden and coauthors [37]:

Bioassay test on brine shrimp, Artemia salina

Brineshrimp medium (BSM) described by Panigrahi and Dallin [38] with slight modification was used. This was prepared by using 30 g sodium chloride; 0.3 g calcium chloride dehydrate; 1.6 g magnesium chloride hexahydrate; 0.5 g magnesium sulphate heptahydrate; 0.8 g potassium chloride; and 6.0 g glycine. The BSM was autoclaved at 121°C for 15 min stored in a brown colored Scott bottle.

Toxicity of each detectable BEA to brine shrimp larvae (Artemia salina L.) was determined. Dried A. salina eggs were hatched in BSM prepared for 24 hours at 27°C in a small water tank provided with the aeration from the motor. 30 mature larvae were selected and transferred into 24-well cell culture plates by exposing it with 5 µl of mycotoxin extract and sterile BSM was added until 500 µl. The test performed in triplicate for BEA extracted against BSM and methanol as a control. Numbers of dead larvae were counted in each dish after 24 hours of incubation at 27°C. Surviving larvae were killed by freezing at -20°C for 12 hours.

Statistical analysis

The toxicity of BEA from F. sacchari and F. subglutinans on adult were studied on A. salina (Brine shrimp medium). One treatment was carried after 24 hours, data were subjected to analysis of variance test (95% confidence level) using independent samples T-test mean values are presented. The mortality was recorded in the control afterward, for LC50, data were analyzed by analysis of variance T-test (Spss 20.0 version).

RESULTS

Identification of F. subglutinans and F. sacchari

A total of 55 different isolates grew from Fusarium Laboratory consisting of F. sacchari and F. subglutinans which was the causal agent of Pokkahboeng disease in sugarcane. Observation of pigmentation, macroscopic and microscopic characteristics were done after cultured on PDA and CLA. This media culture is mainly used for identification purposes as recommended by Leslie and coauthors [2].

The colony morphology of F. sacchari showed abundant mycelia growth after 7 days of incubation on PDA (Figure 1-A). In addition, the pigmentation produced in various colour from pale violet, pink, and peach (Figure 1-B). The observation on CLA, macroconidia usually had 3-septation with slightly falcate and thinwalled (Figure 2 A). The apical cells curved while the basal cells were poorly developed. Microconidia with oval shape together with 1-septate mesoconidia (Figure 2, (A) and (B)) and simple polyphialides were observed on the prepared slide (Figure 3) Mesoconidia and microconidia in situ were present on CLA (Figure 4, (A) and (B)). Chlamydospores and microconidia chains were absent.

For F. subglutinans, the mycelia were abundantly growing on PDA whereas the pigmentations ranging from pale violet to deep violet (Figure 5). From in situ observation on CLA, macroconidia were sparsely formed, slender, and thin-walled with 3- or 4- septa (Figure 6 A). Curved apical cells and poorly developed basal foot-shaped cells were recorded. Oval shaped microconidia with 0-septate (Figure 6 B and 7 A) were found from agar plates attached to monophialides or polyphialides (Figure 7). No chlamydospores or microconidia chains were observing in CLA.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 1

Figure 1. Colony appearance and pigmentation of Fusariumsacchari on PDA. A) Upper surface, B) lower surface.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 2

Figure 2. Microscopic characteristics of Fusariumsacchari, A) Macroconidia, B) Mesoconidia, C) Microconidia.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 3

Figure 3. Simple polyphialidicconidiophores of Fusariumsacchari aerial mycelium

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 4

Figure 4. Polyphialides of Fusarium sacchari on CLA. A) Mesoconidia, B) Microconidia

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 5

Figure 5. Colony appearance and pigmentation of Fusariumsubglutinans on PDA. A) Upper surface. B) Lower Surface.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 6

Figure 6. Microscopic characteristics of Fusariumsubglutinans, A) Macroconidia, B) Microconidia.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 7

Figure 7. Polyphialides of Fusariumsubglutinans on CLA. A) Microconidia, B) False head.

Detection of BEA by TLC

BEA was extracted after 28 days of the inoculation process with F. sacchari and F. subglutinans spore suspension on corn grit cultures. The results of screening were based on the colour spot and comparison of the retention factor (Rf) values of the samples together with the standard of BEA at a concentration of 200 ppm and further visualized under iodine vapour as shown in the Figure (8).

From Table 1, data showed that all the tested isolates of F. sacchari (46) and F. subglutinans (9) having the capable to produce BEA with the TLC method. The visualisedcolour spot of the standard with aid of iodine vapour was brown in colour and the Rf values ranging from 0.93 till 0.97 (Mean value = 0.95). Samples extracted lies almost at the same position with BEA standard colour spots were compared to verify the presence of BEA. Briefly, all isolates of F. sacchari and F. subglutinans showed Rf value in reference to standard BEA. While the colour spots of all isolates appeared resemblance in terms with BEA standard but it is different in the colour intensity. The intensity of colour spots for all isolates was darker compared to the BEA standard exhibited brownish in colour.

Table 1. Beauvericindetection of Fusarium sacchari and Fusarium subglutinans strains isolated from Pokkahboeng disease of sugarcane.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 8

Brine shrimp bioassay

The extracts containing BEA were used to evaluate the toxicity of BEA towards A. salina (Brine shrimp larvae). Two species of the Fusarium in section Liseolais known as F. sacchari and F. subglutinans exhibited a mortality rate up to 100%. Yet each of the isolates of F. sacchari and F. subglutinans was observed to show different mortality rate on A. salina larvae. The results tabulated in Table 2 with the toxicity level indication [39]. All the toxicity levels exhibited varying results ranging from slightly toxic to highly toxic. The calculated t (1.86) was larger than tabulated t_0.05 (1.65). In conclusion, the toxicity of BEA extracts from F. subglutinans was more in comparison to the toxicity of BEA extracts from F. sacchari.

BEA toxicity towards brine shrimp larvae was highly variable. In F. sacchari alone, the mortality rates of brine shrimp towards extracts containing BEA was ranging from 15.56% to 100% as shown in Table 2. The distribution in scale from slightly toxic to very toxic was 5 isolates in the slightly toxic group, 20 isolates in the toxic group, and 21 isolates in the very toxic group. Among those in a very toxic group, 9 isolates showed a 100% mortality rate on brine shrimp larvae.

While BEA extracted from corn grits inoculated with F. subglutinans had a mortality rate in between 66.67-100%. The K3324U isolate of F. subglutinans showed 100% mortality for brine shrimp larvae. Narrow variations in extracts containing BEA extracted from F. subglutinans were more consistent in the BEA production.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 9

Figure 8. Thin layer chromatograms of Beauvericin developed in acetic acid; methanol: water (100:5:1) solvent system. Brown colour spots on silica gel sheet of Beauvericin standard and samples after visualization under iodine vapour. (Lane 1: K3266U, Lane 2: K3247U; Lane 3: K3324U, Lane 4: standard Beauvericin, Lane 5: K3260U, Lane 6: K3311U and Lane 7: K3350U.

Table 2. Beauvericindetection of Fusarium sacchari and Fusarium subglutinans strains isolated from Pokkahboeng disease of sugarcane.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane - Image 10

DISCUSSION

The media PDA and CLA were used instead of others as these media allowed pigmentation of Fusarium and the production of conidia for identification purposes. Fisher and coauthors [32] reported that CLA is a natural substrate medium that promoted sporulation rather than mycelia growth. Microconidia chains, false heads, polyphialides, monophialides, chlamydospores, and sporodochia can be seen in situ if these structures are present.The results showed no difference between F. sacchariand F. subglutinansin producing microconidia, and shape of macroconidia. Morphological characteristics are possible to discriminate into the two species based on the presence of mesoconidia. The distinguishable trait in producing of mesoconidia showed the ability of F. sacchari in producing a higher number of mesoconidia compared with F. subglutinans in situ on CLA media.

Interestingly, the results showed the ability of F. sacchari and F. subglutinans to produce BEA by using the TLC method. At detectable levels on the TLC method after visualization under iodine vapour. They assumed the mycotoxin profile of F. sacchari similar to those of F. subglutinans[2]. Therefore, this assumption was well supported in this study. BEA is a nonpolar cyclodepsipeptide compound as it gives high Rf value due to weak interaction with the polar adsorbent on the TLC plate [40]. The Rf value 0.95 detected in the experiment of BEA was in agreement with previous studies with minor changes in Rf value due to the diverse developing solvent system. The solvent system made up of methanol, water, and formic acid (30:45:25) showed the Rf value of BEA = 0.90 [41]. While, the BEA Rf value under the solvent system of 1-butanol, water, and acetic acid (12:5:3) was 0.92 [40]. The results showed slight dissimilarity in BEA Rf value compared with the previous studies due to the different solvent systems. The difference in the Rf value might be due to several factors including the amount of material spotted, temperature, solvent system, and chemical nature of absorbent. The condition of temperature, the equilibrium between liquids and vapour in tank, sample impurities are the key to a high range of Rf value [42-44]. In addition, the results showed a difference in intensity of colour spots between BEA that extracted from all isolates and BEA standards. The darker brown colour denoted a higher concentration for BEA which was more than 200 ppm contained in the sample extraction [45].

However, the crude extracts for all F. sacchari isolates in this study tested positive for the production of BEA. Moretti and coauthors [25] detected the ability of F. sacchari to produce BEA. Petrovic and coauthors [46] mentioned about F. sacchari produced BEA and a plant pathogen for sugarcane and other crops. F. subglutinans is considered the producer for BEA. This study showed the ability of all tropical isolates of F. subglutinans for producing BEA. Moretti and coauthors [24], Moretti and caouthors [47], and Reyes-Velázquez and coauthors [48] found some isolates of F. subglutinans of different geographic areas could not produce BEA. Several Fusarium sp. produced a BEA toxin including F. verticillioides, F. oxysporum, F. poae¸ F. redolens, F. proliferatum, and F. subglutinans [20,35,48-51].

On the other hand, the result of a biotoxicity assay for the biological activity could provide the detection of either known or unknown mycotoxin in foodstuffs and useful for verification about the toxin presence after screened through chemical means. Hamill and coauthors [52] further confirmed brine shrimp as a sensitive organism towards BEA, so it was selected as the targeted organism in this bioassay test for this study. The study was focus on the mortality rate of brine shrimp towards BEA that calculated to determine the toxicity of BEA. Bioassay test confirmed by many certain studies including Logriecoand coauthors[53], Moretti and caouthors[24], and Moretti and coauthors[25], that calculated the mortality rate of brine shrimp to resolve the toxicity of mycotoxins. Indeed, BEA of F. sacchari and F. subglutinans showed a toxicity for the brine shrimp in the different levels among the same species. Two species of Fusarium in this study is shown three levels of toxicity. Butt and Goettel[54] reported the role of fungal toxins causing death for the host tissue by the combination of colonizing of fungal with nutrient depletion and the action of toxins from fungal. The toxicity BEA levels of F. sacchari included 46% high toxic, 43% toxic, and 11% slightly toxic of isolates. As well, the BEA toxicity of F. subglutinans appeared two groups included 44% high toxicity, and 56% toxic of isolates. The difference in the production BEA among the same species of Fusarium could occur in the wild population of Fusarium spp. because it may have happened a mutation. The change in production of mycotoxins resulted from the effect of a mutation on plant pathogens [55].The mutation in the Fusarium isolates may occur as a result of several factors; (1) the interaction between Fusarium and the host plant such as influence of plant defences,(2) the blend in mating occurred among avirulence and high virulence (56),(3) the type of nutrient, the preservation conditions, and the length of preservation time.

CONCLUSION

The difference in toxicity for the brine shrimp reflected the difference in the capability of Fusarium isolates to cause the pathogenicity. This study manifests the capability of both F. sacchari and F. subglutinans to produce effective BEA. Therefore, BEA may play a big role in the occurrence of the infection for the host plant by causing the toxicity for the host tissues and can be a potential pathogenicity factor.

This article was originally published in Brazilian Archives of Biology and Technology. Vol.64: e21200088, 2021. https://doi.org/10.1590/1678-4324-2021200088. This is an Open Access article under the terms and conditions of the Creative Commons Attribution (CC BY NC) license (https://creativecommons.org/licenses/by-nc/4.0/).

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane

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