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2023 Volume 3
Article Contents
Abstract
Introduction
Materials and methods
Materials
Preparation of mixed surimi samples
Rheological characteristics of mixed surimi samples
Apparent viscosity
Dynamic rheological behavior
Strength of mixed surimi gels
Textural properties of mixed surimi gels
Cooking loss of mixed surimi gels
Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels
Color difference of mixed surimi gels
Scanning electron microscopy (SEM) of mixed surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Statistical analysis
Results and discussion
Rheological properties of surimi gels
Steady-state shear properties
Dynamic elastic properties during gelation
Strength of surimi gels
Textural properties of surimi gels
Cooking loss of mixed surimi gels
Color of surimi gels
Changes in moisture distribution of surimi gels
Scanning electron microscopy of surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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ARTICLE   Open Access    

Toxicity of fusaric acid and beauvericin in tissue-cultured banana 'Grand Nain' and 'Lakatan'

  • 1.

    Institute of Plant Breeding, College of Agriculture and Food Science, University of the Philippines, Los Baños, Laguna, Philippines 4031

  • 2.

    Department of Biological Sciences and Environmental Studies, College of Science and Mathematics, University of the Philippines Mindanao, Philippines 8022

  • 3.

    Department of Biology, College of Science, De La Salle University, Manila, Philippines 1004

More Information
  • Fusarium oxysporum forma specialis cubense (Foc) produces toxins known to contribute to virulence and infection in the host. Fusaric acid (FA) and beauvericin (BEA) are major toxins contributing to Foc virulence in the host plant. Recent advancements allow the production of disease-resistant crops via cell selection, a process that involves resistance screening of somaclones using phytotoxin exposure. Determination of the appropriate concentration is an important step for the toxin-based selection of disease-resistant plants. In this study, the toxicity of FA (0, 5, 10, 20, 30, 50, and 100 μm) and BEA (0, 5, 10, and 20 μm) in different tissues of banana cv. ‘Grand Nain’ and 'Lakatan' were investigated. Overall results indicated a positive relationship between the toxin concentration and plant sensitivity, as indicated by the degree of vascular browning rate. Results demonstrated that lower concentrations of BEA are required for phytotoxicity than in FA. Furthermore, a higher degree of vascular browning was recorded in the test tissues of ‘Lakatan’ treated with FA and BEA than in the ‘Grand Nain’ genotype suggesting genotype-dependent sensitivity of banana to phytotoxins. To our knowledge, this study is the first to investigate the phytotoxicity of FA and BEA in callus cultures of banana ‘Lakatan.’

  • Introduction

    Surimi gel, known as 'concentrated myofibrillar protein'[1], is a kind of gel prepared by processing fish tissue according to fixed steps, such as rinsing, dehydration, and chopping, then adding a certain number of auxiliary materials for crushing, molding, heating and cooling. Salt-soluble myofibrillar protein (mainly myosin) of surimi denatures and unfolds after heating, and then re-crosslinks and polymerizes to form large protein aggregates[2], which is the internal mechanism of forming the gel structure. To enhance the texture and taste of surimi gel products, 2%−3% salt is added to promote the formation of the protein gel network structure and enhance the solubility and functional properties of the products[3,4]. Nonetheless, numerous studies have confirmed that excessive salt intake may result in risks of disease to human health, such as hypertension, and coronary heart disease[5]. Therefore, the development of low-salt surimi products will be widely focused on in future research.

    Recently, two strategies have been innovatively proposed to improve or maintain the gel properties of low-salt surimi products: one is to use exogenous additives or sodium salt substitutes[6−8], and the other is to exploit new processing technologies[9−11]. Glutamine transaminase (TGase) is the most effective surimi quality improver to enhance the functional properties of protein, which could catalyze acyl donors in proteins (γ-Hydroxylamine group) and acyl receptors (lysine residue, primary amine compound, etc.) undergo acyl transfer reaction to form cross-linked structures[12−14]. Previous studies by Jiang et al.[15] revealed that the cross-linking effect of TGase catalysis depends on the content and spatial distribution of available substrates. In addition, lysine, as an ideal exogenous additive, has been widely used in different meat protein gel systems, which can effectively improve the gel properties of low-salt surimi gel[16] and emulsified chicken sausage[17]. Many researchers have spent a lot of time to obtain higher-quality surimi gel products through the combination of two or more exogenous additives. Many scientists devoted themselves to obtaining higher quality surimi gel products through the combination of two or more exogenous additives. For example, Cao et al.[18] demonstrated that lysine (Lys) could bring about the dissociation of actin under the condition of the presence of TGase, promoting the cooking yield and gel properties of oxidative damaged MP. Similar findings reported by Cando et al.[19] indicated that Lys could induce the changes in protein structure, which were favorable terms to heighten the cross-linking effect of TG and improve the strength of surimi gel.

    Recently, the term ε-Poly-lysine (ε-PL) was followed with interest since it's a natural amino acid polymer produced by microbial fermentation, and was speculated that it has a similar molecular structure and potential effect to Lys[20]. ε-PL, which is generally composed of 25−30 lysine residues connected by amide bonds (by ε-Amino and α-Carboxyl group), was often used as an antibacterial agent for the preservation of meat products and aquatic products[21]. Li et al.[21] explored the effect of preservative coating with ε-PL on the quality of sea bass fillets during storage. It turned out that, ε-PL treatment evidently reduced the thiobarbituric acid and volatile base nitrogen values of bass slices during storage, inhibited the growth of microorganisms, and improved the water retention, texture, and flavor characteristics of the fish. A previous study by Cai et al.[20] proved the bacteriostatic and fresh-keeping effect of the composite coating of protein and sodium alginate on Japanese sea bass, and found that the composite coating had an effect on inhibiting the proliferation of sea bass microorganisms (Escherichia coli, lactic acid bacteria, yeast, etc.), fat oxidation, protein degradation, and nucleotide decomposition. As a natural cationic polypeptide, ε-PL has the advantages of no biological toxicity and low viscosity of aqueous solution. It is noteworthy that it can produce strong electrostatic adsorption with negatively charged amino acids[22]. Its effective modification of protein helps it fully interact with gel component molecules and groups[23], which may play a role in enhancing the texture and water retention of protein gel.

    Nevertheless, few reports are based on the addition of ε-PL to explore the influence mechanism on TGase-catalyzed surimi gel properties. The current study sought to shed light on the effects of different additions of ε-PL on TGase-induced cross-linking effect and the performance of composite surimi gel, to provide a theoretical basis and reference for further research and development of 'low salt', efficient and healthy surimi products.

    Materials and methods

    Materials

    Testing materials, including marine surimi, transglutaminase (TG, enzyme activity 100 IU/g), and ε-Polylysine (ε-PL, ≥ 99% purity) were supplied by Jiangsu Yiming Biological Technology Co., Ltd (Jiangsu, China). Egg white protein was purchased from Henan Wanbang Chemical Technology Co., Ltd (Henan, China). All other chemicals were from Shanghai Yuanye Biochemical Co., Ltd (Shanghai, China) and were at least analytical grade. The ingredients of the surimi gel are shown in Table 1.

    Table 1.  Surimi samples with different treatments.
    GroupsSurimi
    (g)    		TGase
    (w/w, %)    		Egg white
    Protein
    (w/w, %)    		ε-PL
    (w/w, %)    		NaCl
    (w/w, %)
    CK300−−−0.5
    TE3000.47.0−0.5
    TE + P13000.47.00.0050.5
    TE + P23000.47.00.010.5
    TE + P33000.47.00.020.5
    TE + P43000.47.00.040.5
    TE + P53000.47.00.060.5
     | Show Table
    DownLoad: CSV

    Preparation of mixed surimi samples

    The prepared block surimi was firstly put into the chopping machine (or tissue masher) and chopped for 2 min, then different mass fractions of ingredients were added, while the chopping time was extended to 5 min (the temperature should be controlled below 10 °C during this process). The exhausted surimi paste was poured into the special mold (50 mm × 20 mm), which was heated in two stages to form the heat-induced surimi gel (40 °C water bath for 40 min; 90 °C water bath for 20 min). Above prepared surimi gel underwent an ice water bath for 30 min and finally stored overnight at 4 °C for further use.

    Rheological characteristics of mixed surimi samples

    Mixed surimi samples with different treatments were determined under a Haake Mars 60 Rheometer (Thermo Fisher Scientific, Germany) with 35 mm stainless steel parallel plates referring to the method described by Cao et al.[18], and each determination was repeated three times. After centrifugation (1,000× g, 3 min) and 4 °C, the degassed surimi sol (~2 g) was equilibrated at 4 °C for 3 min before measurement.

    Apparent viscosity

    The shear stress of the mixed surimi sol was measured at shear rates between 0−100 s−1.

    Dynamic rheological behavior

    The rheological properties of the mixed surimi sols were measured in an oscillatory mode of CD-Auto Strain at 0.02% and 0.1 Hz frequency, respectively. The heating temperature range is set to 20~90 °C while the heating rate is 1 °C·min−1.

    Strength of mixed surimi gels

    The TA-XT Plus physical property analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) was used to analyze the mixed surimi gel strength of each group of samples (n ≥ 3) at room temperature, and the cube-shaped surimi gel samples are measured through the P/0.5 probe. Referring to the method previously described by Fang et al.[24], the test parameters are as follows, pre-test and test rate (1 mm·s−1); rate after measurement (5 mm·s−1); depressing degree (30%); trigger force (5 g); data acquisition rate (400 p/s).

    Textural properties of mixed surimi gels

    Concerning the method of Jirawat et al.[25], texture profile analysis (TPA) was employed to record the hardness, elasticity, cohesion, chewiness, and resilience of mixed surimi gels. It is well known that TPA can explore the textural properties of food through the texture analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) equipped with a P/75 probe to simulate human oral chewing action and obtain the texture characteristic values related to human sensory evaluation. The physical property parameter settings were as follows: downforce (5 g), compression degree (50%), pre-test speed, test speed, and post-test speed (1.0 mm·s−1).

    Cooking loss of mixed surimi gels

    The cooking loss of surimi gel under different treatments was determined referring to the protocol of Dong et al.[26]. The cooked surimi gel sample was instantly absorbed dry and weighed (W5). The cooking loss (CL) is calculated as follows:

    CL(g/100g)=(M1−M2)/M1×100 (1)

    Where M1, weight of the sample before cooking; M2, weight of the sample after cooking.

    Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels

    The LF-NMR of the mixed surimi gel was detected by a PQ001-20-025V NMR analyzer (Niumag Analytical Instruments Co., Ltd, Suzhou, China) referring to the previous method conducted by Li et al.[27]. Relevant characteristic parameters were set as follows: sampling frequency (200 KHZ), echo time (0.3 ms), and cumulative number (8). Keeping the surimi gel sample (approximately 2 g) at room temperature for 30 min, they were carefully put into a cylindrical nuclear magnetic tube (15 mm in diameter), and finally, the relaxation time (T2) was recorded using the Carr Purcell Meiboom Gill (CPMG) pulse sequence.

    Color difference of mixed surimi gels

    CM-5 colorimeter (Konica Minolta Sensing, Inc., Tokyo, Japan) was used for surimi gel with different treatments following to the procedure of Wang et al.[28]. Several 1 cm thick slices were cut from surimi samples (n = 3) selected from different treatment groups, and the gel whiteness was calculated according to the following formula:

    Whiteness=100−√(100−L∗)2+a∗2+b∗2 (2)

    Scanning electron microscopy (SEM) of mixed surimi gels

    According to the previously described procedure by Gao et al.[29], the square-shaped surimi gels (4 mm × 4 mm × 4 mm) were immersed in 0.1 mol·L−1 phosphate buffer (pH 7.2) containing 2.5% (v/v) glutaraldehyde for 24 h. The above samples were washed using phosphate buffer (0.1 mol·L−1, pH 7.2) three times and then subsequently dehydrated in a series of alcohol solutions. The microstructures of the mixed surimi gels were imaged using an FEI Verios 460 SEM (FEI Inc., Hillsboro, OR, USA).

    Thiobarbituric acid-reactive substances (TBARS) determination

    The TBARS value was analyzed by the method described by Hu et al.[30] with slight modification. Thiobarbituric acid solution (1.5 mL) and 8.5 mL of trichloroacetic acid solution were added to the sample in turn, the mixture was bathed in water at 100 °C for 30 min. The supernatant was extracted and centrifuged twice (3,000× g, 5 min): (1) The original supernatant (5 mL) was taken and the same amount of chloroform for mixed centrifugation; (2) The second supernatant (3 mL) and petroleum ether (1.5 mL) were taken for mixed centrifugation. Finally, a small amount of lower liquid was taken to determine the absorbance (A532 nm). The final result was expressed in malondialdehyde equivalent (mg·kg−1).

    Statistical analysis

    All statistical analyses of data were investigated by statistical product and service solutions IBM SPSS Statistics version 23.0 (IBM SPSS Inc., Chicago, USA). The LSD all-pairwise multiple comparison method was used for the least significance analysis, and p < 0.05 was considered to indicate significance. The experimental data are expressed as mean ± standard deviation (SD) and plotted using Origin 2019 (Origin Lab, Northampton, MA, USA) software.

    Results and discussion

    Rheological properties of surimi gels

    Steady-state shear properties

    The steady-state shear flow curve can be used to characterize the interaction between proteins. The steady shear flow changes of apparent viscosity (Pa·s) of composite surimi with shear rate (s−1) under different treatments are shown in Fig. 1a. The apparent viscosity of all samples decreased significantly (p < 0.05) with increasing shear rate, exhibiting shear-thinning behavior[31]. In the range of shear rates from 0.1 to 100 s−1, the samples with TGase addition were always higher than the control, indicating that the induction of TGase enhanced the cross-linking of surimi proteins and formed a more stable structure. The surimi samples added with a lower proportion of ε-PL (0.005% and 0.01%) were regarded as lower viscosity fluid (Fig. 1a). The possible reason was that the low concentration of ε-PL provided a weak effect on the pH value of the surimi system. As a consequence, the content of the net charge provided was relatively small and the electrostatic interaction between the surimi proteins was weakened[20].

    Figure 1.  Effect of different treatments on rheological properties of surimi.
    DownLoad: Full-Size Img PowerPoint
    Dynamic elastic properties during gelation

    The elastic modulus (G′) mainly depends on the interaction between protein molecules, which can reflect the change of elasticity in the heat-induced surimi gel. As shown in Fig. 1b, the gel process of surimi in the control group was a thermodynamic process consisting of two typical stages. In the first stage, the value of G' showed a gradual downward trend in the range of 20~50 °C. This is mainly because, (1) the activity of endogenous protease in surimi is increased; (2) the myosin light chain subunits of surimi are dissociated under the action of protease, forming myosin and actin[24]; (3) heat-induced hydrogen bonds break between protein molecules, thus enhancing the fluidity of the gel system[31]. The second stage: From 50 to 73 °C, the G' value increases rapidly. As the temperature continues to rise to 90 °C, the G 'value keeps rising steadily. At this time, the gel network structure gradually became stable and irreversible.

    Compared with several curves in Fig. 1b, TGase treatment could significantly increase the G' value. The change trend of G' value of the samples added with TGase and ε-PL were similar to that of the control group, but the former increased faster and the maximum value of G' was also significantly higher. The temperature corresponding to the first peak of G' gradually decreased (PL1≈PL2 < PL4≈TE < PL3≈PL5 < CK), indicating that the process of protein denaturation and aggregation was advanced. This result showed that the proper amount of ε-PL (0.04%) in combination with TGase can reduce the thermal denaturation temperature of protein-forming gel and improve the forming ability of composite gel, which was unanimous with the changes in texture and properties of surimi gel (Table 2). Furthermore, the addition of a small amount (0.005%) or an excessive amount (0.06%) of ε-PL made the mixed surimi protein system more unstable, affecting the ability of TG-induced gel formation as the last resort[32].

    Table 2.  Effect of different treatments on the textural properties of surimi gels.
    GroupsHardness (g)SpringinessCohesivenessChewiness (g)Resilience
    CK811.40 ± 45.28de0.81 ± 0.01a0.53 ± 0.02b498.36 ± 21.58c0.21 ± 0.01e
    TE1105.80 ± 61.83c0.83 ± 0.01a0.56 ± 0.01b510.40 ± 26.04c0.24 ± 0.00bcd
    TE + P1736.01 ± 18.49e0.80 ± 0.03a0.61 ± 0.00a348.78 ± 18.36d0.23 ± 0.01cde
    TE + P2777.45 ± 39.61de0.80 ± 0.01a0.60 ± 0.02a307.03 ± 12.97e0.22 ± 0.00de
    TE + P3869.66 ± 45.82d0.83 ± 0.02a0.63 ± 0.01a485.06 ± 22.73c0.28 ± 0.00a
    TE + P41492.80 ± 77.13a0.83 ± 0.01a0.60 ± 0.01a721.74 ± 31.55a0.26 ± 0.01ab
    TE + P51314.60 ± 84.10b0.81 ± 0.00a0.60 ± 0.02a652.36 ± 30.24b0.25 ± 0.01bc
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Strength of surimi gels

    Gel strength is one of the vital indicators to test the quality of surimi products, which directly affects the texture characteristics and sensory acceptance of the products. The strength of mixed surimi gel with TGase was significantly enhanced by 23.97% (p < 0.05) compared with the control group after heat-inducing. This enhancement is probably caused by the crosslinking promotion of TGase (Fig. 2a). A deeper explanation is that under the catalysis of TGase, the ε-amino group on lysine and the γ-amide group on glutamic acid residues undergo acylation reaction inside or between proteins, forming ε-(γ-Glutamyl)-lysine covalent cross-linking bond which promotes the production of the protein gel network[26].

    Figure 2.  (a) Catalytic reaction of glutamine transaminase and (b) effect of different treatments on gel strength and cooking loss of surimi gel. a−d/A−D: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    On the basis of adding TGase, the strength of the mixed gel with ε-PL concentration ranging from 0.005% to 0.03% was lower than that of TE (Fig. 2b). It was speculated that the surimi sample with low concentration ε-PL was a fluid with lower viscosity, and the interaction between protein and water is strengthened, while the electrostatic interaction between proteins is further weakened[33]. This was also consistent with the change in rheological properties in Fig. 1a. Nevertheless, with the increase of ε-PL content to 0.04%, the gel strength of surimi gel significantly increased and reached the highest value (781.63 g·cm), which was about 21.03% higher than that of the TE group (p < 0.05) (Fig. 2b). The increase of gel strength in this process was obtained due to the following three possible reasons, (1) ε-PL is a cationic amino acid with positive charge, which can improve the pH value of protein or meat product system; (2) The interaction between the ε-amino group of ε-PL and the aromatic residue of protein, namely the cation-π interaction, can change the structure of meat protein[34]; (3) Protein (mixed surimi system) with high ε-amino group content, TGase has strong gel improving ability[35].

    Such synergy (ε-PL = 0.04%), as a comprehensive result of different impactors, is that after the surimi was mixed under the optimal ratio conditions, the synthesis of TGase enzyme catalysis, pH value shift, changes in protein and amino acid composition, etc., increased the strength of various forces, thus forming a denser stereoscopic network structure. Note that when the content of ε-PL reached 0.06%, the gel strength of the corresponding surimi gel decreased by 10% compared with 0.04%. Similarly, others reported the result that alkaline amino acids led to the reduction of the strength of the myosin gel of bighead carp[36].

    Textural properties of surimi gels

    TPA, also known as whole texture, is a comprehensive parameter that determines the sensory quality of surimi gel, including hardness, elasticity, cohesion, chewiness, and resilience[37]. As shown in Table 2, the addition of TGase significantly improved the texture properties of surimi gel (p < 0.05), which is related to TGase's ability to induce surimi protein to form more ε-(γ-Glu)-Lys covalent bond during heating[38] (Fig. 2a). Compared with the cross-linking induced by TGase alone, the gel hardness of the mixed surimi gel treated with ε-PL showed a similar change to the gel strength (Fig. 2b). As the concentration of ε-PL increased from 0.005% to 0.04%, the hardness of surimi gel gradually increased until it reached the maximum (1,492.80 g). At the same time, we also observed that the elasticity, cohesion, and chewiness of the composite gel reached the highest values with the 0.04% ε-PL, and these were higher than those of the TGase-only group. Similar to the findings of Ali et al.[39], the author found that the combination of ε-PL and beetroot extract can effectively replace nitrite, which produced a marked effect in maintaining the color of Frankfurt sausage and improving the texture and performance. However, the data we collated above (Table 2) also showed a clear phenomenon, that is, the addition of ε-PL with lower concentration (0.005%−0.02%) or highest concentration (0.06%) was not conducive to the combination with TGase to improve the texture characteristics of mixed surimi gel.

    Cooking loss of mixed surimi gels

    Cooking loss indicates the water holding capacity of heated surimi, which usually represents the stability of the three-dimensional network structure of surimi gel[40]. The 0.4% TGase significantly reduced the cooking loss of surimi samples (p < 0.05), which was 4.91% lower than that of the control group (Fig. 2b). When the addition of PL (combined with TGase) gradually increased from 0.005% to 0.04%, the cooking loss of surimi showed a decreasing trend and reached the minimum at 0.04%. The ε-PL-added surimi gel samples had higher cooking loss compared with that of only with TGase. Most notably, this may be due to ε-PL is able to further exert the cross-linking effect of TGase. A similar finding was reported in the study by Ma et al.[22], adding ε-PL is able to improve the solubility of myofibrillar proteins. Proteins with high solubility are more easily induced by TGase during heating, which aggravates the cross-linking between ε-PL-protein or protein-protein and forms a disordered gel network structure with relatively weaker water-holding capacity.

    Color of surimi gels

    As shown in Table 3, the high lightness (L*), low yellowness (b*), and high whiteness of surimi in the control group were relatively low, with values of 72.49, 10.42, and 70.56 respectively. After TGase was added to induce cross-linking, these values increased significantly (p < 0.05), which may be related to the photochromic effect of water molecules released from gel matrix under TGase-induced surimi protein cross-linking reported in the previous study[41]. Furthermore, on the basis of adding TGase, it was interesting to see that the L* and whiteness values of surimi gel first increased and then gradually decreased with the increase in ε-PL concentration. The influence of ε-PL combined with TGase on the whiteness of gel can be attributed to three reasons: (1) The addition of ε-PL can effectively increase the substrate content of TGase, and promote the cross-linking between proteins, so as to obtain a much more compact surimi gel structure, which affects the refractive index of light; (2) The water retention performance of the high surimi gel was significantly improved with the addition of ε-PL[42] (Fig. 2b), at this time, the L* value (positively related to the whiteness) value showed a decreasing trend with the decrease of the surface free water content of the gel sample; (3) With the increase of ε-PL concentration, some colored substances, formed due to the accelerated Maillard reaction rate during the preparation of gel, may have an adverse effect on the improvement of gel whiteness[43]. After adding TGase to induce cross-linking, although the whiteness value of composite surimi was significantly enhanced, ε-PL was not enough to further improve the whiteness value of final mixed surimi gel, and high concentration so far as to have a negative impact on the whiteness value.

    Table 3.  Effect of different treatments on the color of surimi gels.
    GroupsL*a*b*Whiteness
    CK72.49 ± 0.82e−0.92 ± 0.01e10.42 ± 0.33d70.56 ± 0.65d
    TE77.82 ± 0.34ab−0.23 ± 0.02c12.29 ± 0.29bc74.64 ± 0.26ab
    TE + P178.39 ± 0.40a−0.14 ± 0.01a12.46 ± 0.12ab75.05 ± 0.41a
    TE + P278.42 ± 0.43a−0.17 ± 0.0b12.46 ± 0.15ab75.07 ± 0.32a
    TE + P377.48 ± 0.16b−0.18 ± 0.02b12.37 ± 0.09abc74.31 ± 0.11b
    TE + P476.34 ± 0.05c−0.22 ± 0.01c12.73 ± 0.02a73.13 ± 0.04c
    TE + P575.48 ± 0.18d−0.26 ± 0.01d12.01 ± 0.14c72.70 ± 0.19c
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Changes in moisture distribution of surimi gels

    The water distribution in surimi gel products was measured by LF-NMR[44], and the results were inverted to obtain the transverse relaxation time (T2), which reflects the strength of water fluidity[45]. There are four fitted wave peaks which are assigned to four different water distribution states and wave peak ranges (Fig. 3, Table 4): strong bound water T21 (0.1−1 ms), weak bound water T22 (1−10 ms), non-flowing water T23 (10−100 ms) and free water T24 (100−1,000 ms)[46]. At the same time, similar characteristics (the main peak is centered on T23) are possessed by the water distribution of all surimi gel samples. The addition of TGase increased the proportion of non-flowing water, accompanying the significant decrease in the content of free water (p < 0.05), compared with the control group. It proved that the addition of TGase brought this phenomenon, namely the bound water in surimi gel was partially transferred to the non-flowing, which also indicated the reduction of cooking loss of surimi samples induced by TGase (Fig. 2). On the basis of adding TGase, the low level of ε-PL (0.005%, 0.01% and 0.02%) inhibited the transition of free water in surimi gel to non-flowing water, in which the proportion of T23 decreased by 2.05%, 2.74%, and 1.42% respectively compared with the surimi gel only with TGase, while T24 increased by 15.76%, 14.17%, and 4.43% respectively. The proportion of P23 in the surimi gel sample (ε-PL = 0.04%) was equivalent to that of the TE group, implying that the addition of appropriate ε-PL was not directly connected with the changes in water distribution in surimi after TGase-induced cross-linking, and slight addition would have a negative impact, which was unanimous with the above research results of cooking loss (Fig. 2a).

    Figure 3.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    DownLoad: Full-Size Img PowerPoint
    Table 4.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    GroupsP21 (%)P22 (%)P23 (%)P24 (%)
    CK2.175 ± 0.186c1.198 ± 0.012e82.863 ± 0.691bc13.764 ± 0.091a
    TE1.938 ± 0.095d2.166 ± 0.043a85.144 ± 1.336a10.752 ± 0.033e
    TE + P12.705 ± 0.067b1.414 ± 0.009d83.435 ± 1.616b12.446 ± 0.105c
    TE + P23.005 ± 0.003a1.845 ± 0.056b82.874 ± 1.739bc12.276 ± 0.108c
    TE + P33.107 ± 0.063a1.715 ± 0.033c83.950 ± 1.587ab11.228 ± 0.086d
    TE + P41.770 ± 0.018d2.099 ± 0.059a85.021 ± 1.756a11.110 ± 0.144d
    TE + P53.121 ± 0.007a1.758 ± 0.107bc82.136 ± 1.091c12.985 ± 0.018b
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Scanning electron microscopy of surimi gels

    The effect of different treatment methods on the microstructure of surimi gel was observed by SEM. As shown in Fig. 4, the microstructure of the control surimi gel (Fig. 4a) was observed to be enormously loose, uneven, and irregular, with large pores, which explained the reason why the control surimi gel had low strength and large cooking loss (Fig. 2b). In addition, the morphological properties of another group of surimi gel (TGase-induced) are appreciably different from the scanning electron micrograph mentioned above (Fig. 4b). Numerous compact and evenly distributed small pores were observed, instead of the loose structure in the control sample, which accounted for the fact that TGase could interact with proteins to form a more uniform and orderly three-dimensional network structure in the process of gel formation[47]. Moreover, similar findings were also reported in the previous studies of Dong et al.[26]. When treated with TGase and ε-PL (from 0.05% to 0.04%), the surface of the surimi gel sample gradually became flat through observation (Fig. 4f). We found that some smaller pores were formed and some larger pores were slightly supplemented visually. Remarkably, when the addition of ε-PL increased to 0.06% (Fig. 4g), the structure of surimi gel was not as uniform as before, and some large and irregular holes appeared obviously. This phenomenon might be related to moderate ε-PL, especially 0.02%, which can promote the cross-linking of TGase with protein and the aggregation/denaturation of different components, which agreed with the change of coagulation strength of surimi samples.

    Figure 4.  Effect of different treatments on the microstructure of surimi.
    DownLoad: Full-Size Img PowerPoint

    Thiobarbituric acid-reactive substances (TBARS) determination

    TBARS value, which can reflect the degree of lipid oxidation and rancidity of surimi, is an important indicator for the occurrence of oxidation[48]. Some substances in surimi, such as unsaturated fatty acids, will inevitably undergo oxidative decomposition to produce a large amount of malondialdehyde (MDA), the latter reacts easily with thiobarbituric acid (TBA) reagent to produce red compounds with maximum absorbance at 532 nm. As illustrated in Fig. 5, when TGase and a certain amount of PL (from 0.005% to 0.06%) were added, the TBARs value of surimi gels decreased to 2.03 and 1.67 mg/kg respectively, compared with the control group (2.26 mg/kg). This shows that the addition of both can significantly reduce the TBARS value of surimi gel (p < 0.05), considered to be a cue for the fat oxidation of surimi being inhibited. In addition, we also found that the antioxidant effect of PL was dose-dependent. The ε-PL can be chelated by ferrous ions along with the ability to scavenge free radicals like hydroxyl radicals, ensuring its antioxidant properties[33]. The ε-PL was prospectively considered as an antioxidant additive to prevent surimi protein from deteriorating induced by oxidation[49]. Similarly, making use of lysine or ε-PL as antioxidants in plant protein and meat protein has been widely reported[33, 50]. Fan et al.[42] confirmed that ε-PL addition has a significant effect on maintaining a high total phenolic content and vitamin C levels in fresh lettuce, which also shows that it has certain antioxidant activity.

    Figure 5.  Effect of different treatments on TBARS values of surimi gel. a−f: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    Conclusions

    The addition of ε-PL at different concentrations had an apparent impact on the characteristics of TGase-induced mixed surimi gel, accompanied by the following situations: The 0.04% ε-PL provided a synergistic effect to promote the aggregation and crosslinking of surimi proteins induced by TGase; The rheology, LF-NMR and SEM results showed that the appropriate concentration (0.04%) of ε-PL apparently enhanced the initial apparent viscosity and elasticity of surimi samples, which was conducive to the formation of a more dense and uniform three-dimensional network structure, further limiting the flow of water in surimi and the exudation of hydrophilic substances; Simultaneously, the network strength was strengthened along with the texture properties of the mixed surimi gel. Furthermore, ε-PL had a strong ability to inhibit lipid oxidation in mixed surimi gel, showing a concentration dependence. When the content of ε-PL ran to lowest (0.005%−0.01%) or highest (0.6%), the improvement of the quality of surimi mixed gel was opposite to the above. The results show that ε-PL can be perceived as a prospective polyfunctional food additive to improve the texture properties, nutritional advantages, and product stability of surimi products.

  • The authors confirm contribution to the paper as follows: study conception and design: Cao Y, Xiong YL, Yuan F, Liang G; data collection: Liang G, Cao Y, Li Z, Liu M; analysis and interpretation of results: Li Z, Cao Y, Liang G, Liu Z, Liu M; draft manuscript preparation: Li Z, Cao Y, Liu ZL. All authors reviewed the results and approved the final version of the manuscript.

  • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    • Acknowledgments

    This work was financially supported by the Natural Science Basic Research Program of Shaanxi (No. 2023-JC-YB-146), the fund of Cultivation Project of Double First-Class Disciplines of Food Science and Engineering, Beijing Technology & Business University (No. BTBUKF202215), the Innovation Capability Support Plan of Shaanxi (No. 2023WGZJ-YB-27), the Agricultural Technology Research and Development Project of Xi'an Science and Technology Bureau (No. 22NYYF057), and Jiangsu Yiming Biological Technology Co., Ltd. in China.

  • The authors declare that they have no conflict of interest.

  • [1]

    Beckman CH, Mace ME, Halmos S, McGahan MW. 1961. Physical barriers associated with resistance in Fusarium wilt of bananas. Phytopathology 51:507−15

    Google Scholar

    [2]

    Li C, Zuo C, Deng G, Kuang R, Yang Q, et al. 2013. Contamination of bananas with beauvericin and fusaric acid produced by Fusarium oxysporum f. sp. cubense. PLoS ONE 8:e70226

    doi: 10.1371/journal.pone.0070226

    CrossRef   Google Scholar

    [3]

    Fakhouri W, Walker F, Armbruster W, Buchenauer H. 2003. Detoxification of fusaric acid by a nonpathogenic Colletotrichum sp. Physiological and Molecular Plant Pathology 63:263−69

    doi: 10.1016/j.pmpp.2004.03.004

    CrossRef   Google Scholar

    [4]

    Ding Z, Yang L, Wang G, Guo L, Liu L, et al. 2018. Fusaric acid is a virulence factor of Fusarium oxysporum f. sp. cubense on banana plantlets. Tropical Plant Pathology 43:297−305

    doi: 10.1007/s40858-018-0230-4

    CrossRef   Google Scholar

    [5]

    Liu S, Li J, Zhang Y, Liu N, Viljoen A, et al. 2019. Fusaric acid instigates the invasion of banana by Fusarium oxysporum f. sp. cubense TR4. New Phytologist 225:913−929

    doi: 10.1111/nph.16193

    CrossRef   Google Scholar

    [6]

    Drysdale RB. 1984. The production and significance in phytopathology of toxins produced by species of Fusarium. In The Applied mycology of Fusarium, eds. Moss MO, Smith JE. x, 264 pp. Cambridge: Cambridge University Press. pp. 95–105
    [7]

    Marrè MT, Vergani P, Albergoni FG. 1993. Relationship between fusaric acid uptake and its binding to cell structures by leaves of Egeria densa and its toxic effects on membrane permeability and respiration. Physiological and Molecular Plant Pathology 42:141−57

    doi: 10.1006/pmpp.1993.1012

    CrossRef   Google Scholar

    [8]

    Chakrabarti DK, Ghosal S. 1989. The disease cycle of mango malformation induced by Fusarium moniliforme var. subglutinans and the curative effects of mangiferin-metal chelates. Journal of Phytopathology 125:238−46

    doi: 10.1111/j.1439-0434.1989.tb01065.x

    CrossRef   Google Scholar

    [9]

    Gapillout I, Milat ML, Blein JP. 1995. Effect of fusaric acid on cells from tomato cultivars resistant or susceptible to Fusarium oxysporum f. sp.Lycopersici. European Journal of Plant Pathology 102:127−32

    doi: 10.1007/BF01877099

    CrossRef   Google Scholar

    [10]

    D'Alton A, Etherton B. 1984. Effects of fusaric acid on tomato root hair membrane potentials and ATP Levels 1. Plant Physiology 74:39−42

    doi: 10.1104/pp.74.1.39

    CrossRef   Google Scholar

    [11]

    Kuźniak E. 2001. Effect of fusaric acid on reactive oxygen species and antioxidants in tomato cell cultures. Journal of Phytopathology 149:575−82

    doi: 10.1046/j.1439-0434.2001.00682.x

    CrossRef   Google Scholar

    [12]

    Brown DW, Lee SH, Kim LH, Ryu JG, Lee S, et al. 2015. Identification of a 12-gene fusaric acid biosynthetic gene cluster in Fusarium species through comparative and functional genomics. Molecular Plant-Microbe Interactions 28:319−32

    doi: 10.1094/MPMI-09-14-0264-R

    CrossRef   Google Scholar

    [13]

    López-Díaz C, Rahjoo V, Sulyok M, Ghionna V, Martín-Vicente A, et al. 2018. Fusaric acid contributes to virulence of Fusarium oxysporum on plant and mammalian hosts. Molecular Plant Pathology 19:440−53

    doi: 10.1111/mpp.12536

    CrossRef   Google Scholar

    [14]

    Moretti A, Logrieco A, Bottalico A, Ritieni A, Randazzo G, et al. 1995. Beauvericin production by Fusarium subglutinans from different geographical areas. Mycological Research 99:282−86

    doi: 10.1016/S0953-7562(09)80899-X

    CrossRef   Google Scholar

    [15]

    Hamill RL, Higgens CE, Boaz HE, Gorman M. 1969. The structure of beauvericin, a new depsipeptide antibiotic toxic to Artemia salina. Tetrahedron Letters 49:4255−58

    doi: 10.1016/s0040-4039(01)88668-8

    CrossRef   Google Scholar

    [16]

    Hidaka H, Nagatsu T, Takeya K, Takeuchi T, Suda H. 1969. Fusaric acid, a hypertensive agent produced by fungi. The Journal of Antibiotics 22:228−30

    doi: 10.7164/antibiotics.22.228

    CrossRef   Google Scholar

    [17]

    Lemmens-Gruber R, Rachoy B, Steininger E, Kouri K, Saleh P, et al. 2000. The effect of the Fusarium metabolite beauvericin on electromechanical and physiological properties in isolated smooth and heart muscle preparations of guinea pigs. Mycopathologia 149:5−12

    doi: 10.1023/A:1007293812007

    CrossRef   Google Scholar

    [18]

    Logrieco A, Moretti A, Ritieni A, Caiaffa MF, Macchia L. 2001. Beauvericin: chemistry, biology and significance. In Advances in Microbial Toxin Research and Its Biotechnological Exploitation, ed. Upadhyay RK. Amsterdam, Netherlands: Elsevier Science Publishers. pp 23–30. https://doi.org/10.1007/978-1-4757-4439-2_2
    [19]

    Tomoda H, Huang XH, Cao J, Nishida H, Nagao R, et al. 1992. Inhibition of acylCoA: cholesterol acyltransferase activity by cyclodepsipeptide antibiotics. The Journal of Antibiotics 45:1626−32

    doi: 10.7164/antibiotics.45.1626

    CrossRef   Google Scholar

    [20]

    Gavazzi G, Tonelli C, Todesco G, Arreghini E, Raffaldi F, et al. 1987. Somaclonal variation versus chemically induced mutagenesis in tomato (Lycopersicon esculentum L.). Theoretical and Applied Genetics 74:733−38

    doi: 10.1007/BF00247550

    CrossRef   Google Scholar

    [21]

    Péreau-Leroy P. 1958. Le palmier dattier au Maroc. Paris: Institut français de recherches fruitières outremer, Mission au Maroc. 142 pp.
    [22]

    El Hadarami A, El Idrissi-Tourane A, El Hassni M, Daayf F, El Hadrami I. 2005. Toxin-based in-vitro selection and its potential application to date palm for resistance to the bayoud Fusarium wilt. Comptes Rendus Biologies 328:732−44

    doi: 10.1016/j.crvi.2005.05.007

    CrossRef   Google Scholar

    [23]

    Smith RH, Duncan RR, Bhaskaran S. 1993. In vitro selection and somaclonal variation for crop improvement. In International Crop Science I, eds. Buxton DR, Shibles R, Forsberg RA, Blad BL, Asay KH, et al. United States of America: Crop Science Society of America. pp. 269−632. https://doi.org/10.2135/1993.internationalcropscience.c99
    [24]

    Wilson CR, Tegg RS, Wilson AJ, Luckman GA, Eyles A, et al. 2010. Stable and extreme resistance to common scab of potato obtained through somatic cell selection. Phytopathology 100:460−67

    doi: 10.1094/PHYTO-100-5-0460

    CrossRef   Google Scholar

    [25]

    Esmaiel NM, Al-Doss AA, Barakat MN. 2012. In vitro selection for resistance to Fusarium oxysporum f. sp. dianthi and detection of genetic polymorphism via RAPD analysis in carnation. Journal of Medicinal Plants Research 6:3997−4004

    doi: 10.5897/jmpr12.150

    CrossRef   Google Scholar

    [26]

    Purwati RD, Budi US, Sudarsono S. 2007. Penggunaan asam fusarat dalam seleksi in vitro untuk resistensi abaka terhadap Fusarium oxysporum f.sp. cubense. Industrial Crops Research Journal 13:64−72

    Google Scholar

    [27]

    Matsumoto K, Barbosa ML, Souza LAC, Teixeira JB. 1999. In vitro selection for Fusarium wilt resistance in banana. II. Resistance to culture filtrate of race 1 Fusarium oxysporum f. sp. cubense. Fruits 54:151−57

    Google Scholar

    [28]

    Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15:473−97

    doi: 10.1111/j.1399-3054.1962.tb08052.x

    CrossRef   Google Scholar

    [29]

    Molina AB, Sinohin VO, Fabregar EG, Ramillete EB, Loayan MM, et al. 2016. Field resistance of Cavendish somaclonal variants and local banana cultivars to tropical race 4 of Fusarium wilt in the Philippines. Acta Horticulturae 1114:227−30

    doi: 10.17660/actahortic.2016.1114.31

    CrossRef   Google Scholar

  • Cite this article

    Cruz MA, Alcasid C, Millado CS, Balendres MA. 2023. Toxicity of fusaric acid and beauvericin in tissue-cultured banana 'Grand Nain' and 'Lakatan'. Technology in Horticulture 3:15 doi: 10.48130/TIH-2023-0015
    Cruz MA, Alcasid C, Millado CS, Balendres MA. 2023. Toxicity of fusaric acid and beauvericin in tissue-cultured banana 'Grand Nain' and 'Lakatan'. Technology in Horticulture 3:15 doi: 10.48130/TIH-2023-0015
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Toxicity of fusaric acid and beauvericin in tissue-cultured banana 'Grand Nain' and 'Lakatan'

  • 1.

    Institute of Plant Breeding, College of Agriculture and Food Science, University of the Philippines, Los Baños, Laguna, Philippines 4031

  • 2.

    Department of Biological Sciences and Environmental Studies, College of Science and Mathematics, University of the Philippines Mindanao, Philippines 8022

  • 3.

    Department of Biology, College of Science, De La Salle University, Manila, Philippines 1004

  • Received: 23 April 2023
  • Accepted: 18 May 2023
  • Published online: 04 September 2023
Technology in Horticulture  3,  Article number: 15  (2023)  |  Cite this article

Abstract: Fusarium oxysporum forma specialis cubense (Foc) produces toxins known to contribute to virulence and infection in the host. Fusaric acid (FA) and beauvericin (BEA) are major toxins contributing to Foc virulence in the host plant. Recent advancements allow the production of disease-resistant crops via cell selection, a process that involves resistance screening of somaclones using phytotoxin exposure. Determination of the appropriate concentration is an important step for the toxin-based selection of disease-resistant plants. In this study, the toxicity of FA (0, 5, 10, 20, 30, 50, and 100 μm) and BEA (0, 5, 10, and 20 μm) in different tissues of banana cv. ‘Grand Nain’ and 'Lakatan' were investigated. Overall results indicated a positive relationship between the toxin concentration and plant sensitivity, as indicated by the degree of vascular browning rate. Results demonstrated that lower concentrations of BEA are required for phytotoxicity than in FA. Furthermore, a higher degree of vascular browning was recorded in the test tissues of ‘Lakatan’ treated with FA and BEA than in the ‘Grand Nain’ genotype suggesting genotype-dependent sensitivity of banana to phytotoxins. To our knowledge, this study is the first to investigate the phytotoxicity of FA and BEA in callus cultures of banana ‘Lakatan.’

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    Introduction

    • Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is an economically important disease affecting banana. Wilting results from the restrictive movement of water in the vascular bundles[1]. However, part of the pathogenesis and invasion of plants by Foc can be attributed to toxic metabolites produced by the fungus[2]. Major toxins of Foc include fusaric acid and beauvericin, which act as virulence factors for infection by causing significant browning of vascular tissues and plant necrosis[3−5].

      Fusaric acid (FA) is a non-specific toxin many fungal pathogens produce[6]. Based on previous studies, FA supports disease development through the induction of cell membrane early super polarization[3], H+ pumping, K+ leaking suppression[7], mineral chelation[8] and inhibition of plant defensive enzymes activity, leading to reduced cell viability[9], changes in membrane permeability and potential[7,10], and production of reactive oxygen species[11]. Previously, Brown et al. recorded that lack of FA production did not affect the virulence of F. oxysporum in cacti or F. verticillioides in maize seedlings[12]. In contrast, several studies reported the importance of FA production in the virulence of Foc in banana[2,5,13].

      Meanwhile, beauvericin (BEA) is a secondary metabolite produced by several species from the Fusarium fujikuroi species complex[14] and the entomopathogenic fungus Beauveria bassiana[15]. Although FA and BEA are major toxins of Foc, the two are not considered mycotoxins with significant human, animal, food, and feed safety risks[2]. Still, some studies have documented physiologic disorders in experimental animals and human cell lines treated with FA[16] and BEA[17−19].

      Phytotoxin insensitivity of plant cell lines can be used as a potential marker in breeding programs for early screening of resistance against plant pathogens[20,21]. The approach requires a significant correlation between toxin sensitivity and host susceptibility to the pathogens producing them[22]. Several studies have taken advantage of this via somaclonal cell selection - a technique that utilizes phytotoxins as selective agents for developing resistant clones against economically important diseases such as common scab of potato[23,24]. Production of Fusarium wilt-resistant date palm[22], carnation[25], abaca[26], and banana cv. 'Maca'[27] has also been reported by cell selection. Yet, little attention has been given to the phytotoxicity of Foc metabolites in banana.

      This study determined the phytotoxicity of FA and BEA in leaf tissues, multiple bud clumps, tissue-cultured plantlets, and calli in-vitro. These results would aid in determining the appropriate concentration of the metabolites for somaclonal cell selection.

    Materials and methods

      Toxin preparation and assay

    • The test substances: fusaric acid (≥ 99% purity, CAS No. 536-69-6) and beauvericin (≥ 97% purity, CAS No. 28048-05-5), were obtained from Sigma-Aldrich. The toxins were dissolved in 0.05% methanol to obtain a 10 mM stock solution. The stock solution was then filter-sterilized by passing through Whatmann no.1 filter paper (125 mm size) (CAT no. 1001-125, GE Healthcare Life Sciences) and stored in a 4 °C refrigerator until use.

      Phytotoxicity testing in different tissues of banana

    • Different concentrations of fusaric acid (FA) and beauvericin (BEA) in tissue-cultured Cavendish plantlets, multiple bud clumps (MBC), callus, and attached leaf tissues were determined. FA stock solution was further diluted to 0, 1, 5, 10, 20, 30, 50, 70, 100 μm concentrations by adding desired concentrations in culture medium. Meanwhile, 0, 1, 5, 10, and 20 μm BEA were tested. These concentrations were selected based on the previous findings of Li et al.[2] on the phytotoxicity of FA and BEA in tissue-cultured plantlets of banana.

      For tissue-cultured plantlets tests, tissue-cultured 'Grand Nain' and 'Lakatan' were obtained from the National Plant Genetic Resources Laboratory (NPGRL), IPB, UPLB. We used 'Grand Nain' because it is commercially propagated in Mindanao, where the Foc TR4 is present. 'Lakatan' was also used since it is a local variety commonly grown in the Philippines, especially by small-scale growers. The tissue-cultured materials were cultured and maintained in the laboratory for micropropagation. The plantlets were propagated by cutting a 2-cm segment of tissue containing the pseudostem and apical meristems. The tissues were then cut in half and cultured in a multiplication-inducing medium which consisted of Murashige and Skoog medium[28] + 3 mg·L−1 BAP. Four segments were placed in each glass jar. Cultures were maintained in 14 h light with temperature ranging from 20 ± 5 °C. Cultures were routinely transferred in the same medium every two weeks to prevent browning. One-month-old tissue-cultured plantlets with roots were transferred in test tubes containing 10 ml liquid medium and treated with FA and BEA by pipetting desired concentration in the medium. Plantlets treated with sterile distilled water served as control. Cultures were maintained in 8 h light at ambient temperature (20 ± 5 °C).

      For multiple bud clumps studies, the protocol of Matsumoto et al.[27] was used to produce multiple bud clumps. Rhizome tissues were cut from 'Grand Nain' and 'Lakatan' shoot cultures and were grown in MS medium containing BAP (5 mg·L−1) to initiate multiple bud clump (MBC) formations. Cultures were maintained under the same conditions as previously mentioned. One-month-old bud clumps were harvested from a multiple bud clump and transferred into test tubes containing 10 ml liquid medium. Five individual buds were placed in each tube and treated with FA and BEA as previously described.

      Corm segments (approximately 2 cm in size) were cut from one-month-old shoot cultures of banana cv for callus studies. 'Grand Nain' and 'Lakatan' and inoculated on MS basal medium supplemented with 2,4-dichloro phenoxy acetic acid (1.0 mg·L−1) and myo-inositol (50 mg·L−1). Cultures were stored in dark conditions under a chamber covered with black cloth and incubated at ambient temperature (20 ± 5 °C). Individual calli were harvested eight weeks post-inoculation and transferred into test tubes containing 10 ml liquid medium. Five individual calli were placed in each tube and treated with FA and BEA as previously described.

      For the attached leaf assay, tissue-cultured 'Grand Nain' and 'Lakatan' with multiple shoots were grown in basal MS medium for rooting. After one month, the rooted plantlets were acclimatized and transferred to plastic bags containing sterile soil in the nursery. The plantlets were grown for two months and watered daily. FA and BEA were diluted to the desired concentration by adding sterile distilled water in 1.5 ml Eppendorf tubes. The attached leaves were inoculated by pipetting 20 μl of solution in wounded (pricked using a syringe needle) tissues. The experiment was repeated twice.

      Data collection and statistical analyses

    • The phytotoxicity of fusaric acid (FA) and beauvericin (BEA) in different banana tissues was assessed ten days post-inoculation. Wilting rates were measured ocularly based on the 10-point arbitrary scale produced. A one-way ANOVA was performed to identify significant differences between treatments using Statistical Tool for Agricultural Research (STAR Nebula) with a 95% confidence level.

    Results

    • Wilting of different tissues treated with fusaric acid (FA) and beauvericin (BEA) was observed ten days post-inoculation. Ten-point visual hedonic browning scales were developed based on varying degrees of wilting (Fig. 1a−c). The rating scales were used to measure the degree of phytotoxicity in tissue-cultured plantlets (TC plantlets), multiple bud clumps (MBC), and callus cultures.

      Figure 1. 

      Ten point rating scales produced in this study for measuring degree of wilting in (a) tissue cultured plantlets, (b) calli, and (c) multiple bud clumps, as caused by Fusarium toxins.

      Overall, there is an increasing trend between the toxin concentration and the degree of browning observed (Figs 2−4). For both 'Grand Nain' and 'Lakatan,' the highest browning rates were observed in tissues treated with 100 μm FA and 20 μm BEA (Tables 1 & 2). A higher degree of browning was recorded in 'Lakatan' TC plantlets, MBC, and callus than in 'Grand Nain,' except in TC plantlets treated with 20 μm BEA, where a higher average browning rate score was observed in 'Grand Nain' (9) than in 'Lakatan' (Tables 1 & 2).

      Figure 2. 

      Browning rates of tissue-cultured plantlets, calli, and multiple bud clumps of banana cvs. (a), (b) ‘Grand Nain’ and (c), (d) ‘Lakatan' treated with varying concentrations of FA and BEA at 10 d post-incubation.

      Average browning rates of 'Grand Nain' TC plantlets, MBC, and callus treated with 100 μm FA were 3.4, 7.8, and 9.6, respectively, using the 10-point system (Table 1). Meanwhile, on average, the browning rates of tissue-cultured 'Lakatan' TC plantlets, multiple bud clumps, and calli treated with the same concentration were 6.8, 9.6, and 10 (Table 1). For phytotoxicity tests using BEA, 'Grand Nain' TC plantlets, MBC, and callus scored 9, 8.8, and 7.6, while the same tissues of 'Lakatan' scored 7.0, 9.8, and 8.8, respectively (Table 2).

      Table 1.  Wilting rates of different tissues of banana 10 d after fusaric acid treatment.

      FA (µm)'Grand Nain''Lakatan'
      TC plantletMBCCallusTC plantletMBCCallus
      Control0.8 ± 0.37b0.6 ± 0.24c0.2 ± 0.20d0.4 ± 0.24c0.4 ± 0.24e7.8 ± 0.37ab
      11.8 ± 0.49ab1.2 ± 0.20c1.2 ± 0.20cd1.0 ± 0.55c1.4 ± 0.24e2.4 ± 0.68e
      51.6 ± 0.60ab3.8 ± 0.86bc1.4 ± 0.24cd1.2 ± 0.20c1.8 ± 0.37de3.0 ± 0.45e
      101.4 ± 0.24b4.0 ± 1.48bc2.0 ± 0.00cd0.4 ± 0.24c3.8 ± 0.58cd3.6 ± 0.51de
      201.6 ± 0.40ab4.4 ± 1.50abc2.6 ± 0.24c2.0 ± 0.32c5.6 ± 0.24bc3.8 ± 0.58de
      301.4 ± 0.40b4.0 ± 0.00bc5.2 ± 1.24b2.0 ± 0.00c3.6 ± 0.75cd5.4 ± 0.68cd
      501.6 ± 0.24ab7.3 ± 0.56ab7.2 ± 0.49b4.8 ± 0.49b6.0 ± 0.63b7.0 ± 0.00bc
      1003.4 ± 0.24a7.8 ± 0.92a9.6 ± 0.24a6.8 ± 0.80a10.0 ± 0.00a9.6 ± 0.24a
      Values represent the mean ± SE of five replicates Means in a column with the same letter are not significantly different (p > 0.05).

      Table 2.  Wilting rates of different tissues of banana 10 d after beauvericin treatment.

      BEA
      (µm)      		'Grand Nain''Lakatan'
      TC plantletMBCCallusTC plantletMBCCallus
      Control1.0 ± 0.32d0.6 ± 0.40c0.6 ± 0.24d0.4 ± 0.24d0.0 ± 0.55e0.4 ± 0.00d
      14.0 ± 0.63c1.2 ± 0.20c3.2 ± 0.37c1.6 ± 0.24c7.2 ± 0.55d1.4 ± 0.84c
      54.6 ± 0.75c5.4 ± 0.40b4.6 ± 0.51b2.8 ± 0.20b8.4 ± 0.55c4.6 ± 0.55b
      106.8 ± 0.37b6.0 ± 0.32b6.6 ± 0.24a6.2 ± 0.37a9.2 ± 0.55b6.6 ± 0.45a
      209.0 ± 0.45a8.8 ± 0.37a7.6 ± 0.24a7.0 ± 0.45a9.8 ± 0.84a8.8 ± 0.45a
      Values represent the mean ± SE of five replicates Means in a column with the same letter are not significantly different (p > 0.05).

      Treatment with FA and BEA significantly affected the browning rate compared to the control treatment (Tables 1 & 2). For 'Grand Nain,' the browning rate of TC plantlets and MBC significantly increased at 100 μm and 50 μm concentrations, respectively. At the same time, a significant increase in browning can be observed in callus cultures in as low as 20 μm FA (Fig. 3). FA also caused a significant browning of 'Lakatan' TC plantlets and MBC at 50 and 10 μm concentrations, respectively (Fig. 4). Additionally, 1 μm FA sufficiently caused significant browning in 'Lakatan' callus (Table 2, Fig. 4). BEA, at 1 μm concentration, already caused significant browning for both 'Grand Nain' and 'Lakatan' genotypes (Figs 3 & 4), except in 'Grand Nain' MBC where 5 μm BEA significantly affected the browning rate (Fig. 3). For both 'Grand Nain' and 'Lakatan' attached leaf tissues assay, browning around the inoculated sites were observed with 10 μm and 20 μm FA and BEA (Figs 3 & 4). But more severe symptoms were observed in the 'Lakatan' genotype than in 'Grand Nain' (Figs 3 & 4).

      Figure 3. 

      Toxic effect of FA and BEA in different tissues of banana cv. 'Grand Nain'. Varying concentrations were tested in (a), (d) one-month old tissue-cultured plantlets, (b), (e) multiple buds clumps, (c), (f) callus and (g) attached leaf tissues of two-month old greenhouse plantlets were used. Symptoms were assessed at 10 d post-inoculation.

      Figure 4. 

      Toxic effect of FA and BEA in different tissues of banana cv. 'Lakatan'. (a), (d) Varying concentrations were tested in one-month old tissue-cultured plantlets, (b), (e) multiple bud clumps, (c), (f) callus and (g) attached leaf tissues of two-month old greenhouse plantlets were used. Symptoms were assessed at 10 d post-inoculation.

    Discussion

    • This study demonstrated the phytotoxic effect of varying fusaric acid (FA) and beauvericin (BEA) concentrations in banana 'Grand Nain' and 'Lakatan'. The results were consistent with the previous findings of Li et al.[2], where 100 µm FA and 20 µm BEA sufficiently caused toxicity in tissue-cultured bananas. However, a lower concentration of BEA (1 µm) was found to cause wilting in this study. The arbitrary 10-point visual hedonic scales used in this study could be used to measure the phytotoxicity of Fusarium toxins in the test tissues of banana. A higher degree of vascular browning was recorded in the test tissues of 'Lakatan' treated with FA and BEA than in the 'Grand Nain' genotype. This suggests that banana sensitivity to phytotoxins differs from one genotype to another. In a study conducted by Molina et al., they recorded relatively higher susceptibility and disease incidence of 'Lakatan' in the field than in the 'Grand Nain' genotype[29]. Thus, the sensitivity rate of banana tissues to phytotoxins (FA and BEA) in-vitro may be correlated with its susceptibility to Fusarium wilt disease in the field.

      The degree of browning rates of test plants: tissue-cultured plantlets (TC plantlets), multiple bud clumps (MBC), callus, and attached leaf tissues to FA and BEA varied from one another. Higher susceptibility of callus and MBC than TC plantlets and attached leaf tissues were observed, which indicated higher sensitivity of the tissues to the phytotoxins. This confirms callus and MBC as suitable materials for screening resistant cell lines via somatic cell selection due to heightened toxin sensitivity, although the reason behind this is still unclear. A lower browning rate of attached leaf tissues than in in-vitro test plants demonstrated that toxin sensitivity was more pronounced in vitro than in planta. This may be attributed to several factors, including the environment and the type of tissues[2].

    Conclusions

    • Fusaric acid (FA) and beauvericin (BEA) are toxic to banana 'Grand Nain' and 'Lakatan' through significant vascular browning. The arbitrary 10-point visual hedonic scales developed in this study for tissue-cultured plantlets (TC plantlets), multiple bud clumps (MBC), and callus tissues could be used to measure the phytotoxicity of Fusarium toxins. The higher degree of vascular browning observed in 'Lakatan' banana treated with FA and BEA toxins than in 'Grand Nain' suggested genotype-dependent toxin sensitivity and subsequent susceptibility of banana to Fusarium wilt disease, although further data are needed to support this. Data obtained here would aid in determining the effective concentration for toxin-based cell selection using callus and MBC cultures.

      Acknowledgments

      • This study was supported by the Department of Agriculture- Bureau of Agricultural Research (DA-BAR), Philippines. We thank Fe dela Cueva, Edzel Evallo, Diana Rose Biglete, Monica Fronda, Yron Retuta, Rodel Pia, Pamela Quintos, May Eljera, Loida Pascual, Flora R. Cuevas, Rochelle Delgado, and Eugene Parañaque for the technical assistance and support. Special thanks to the Fruit Crops Section and the National Plant Genetic Resources Laboratory of the Institute of Plant Breeding, College of Agriculture and Food Science for providing the banana cultures and plantlets.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

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      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (4)  Table (2) References (29)
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    Cruz MA, Alcasid C, Millado CS, Balendres MA. 2023. Toxicity of fusaric acid and beauvericin in tissue-cultured banana 'Grand Nain' and 'Lakatan'. Technology in Horticulture 3:15 doi: 10.48130/TIH-2023-0015
    Cruz MA, Alcasid C, Millado CS, Balendres MA. 2023. Toxicity of fusaric acid and beauvericin in tissue-cultured banana 'Grand Nain' and 'Lakatan'. Technology in Horticulture 3:15 doi: 10.48130/TIH-2023-0015
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  • Introduction
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  • Conflict of interest
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