Search
Submit Manuscript
Advanced Search
MAP  >  Technology in Horticulture
2023 Volume 3
Article Contents
Abstract
Introduction
Materials and methods
Plant germplasms, RNA extraction, and DNA extraction
cDNA preparation and Illumina sequencing
Transcriptome assembly and annotation
SSRs identification and primer design
EST-SSR amplification
Genotyping and data analysis
Results
Sequencing and de novo assembly
Annotation and classification of Phalaris transcripts
Prediction of repeat motif types
Development and polymorphism identification of EST-SSR markers
Clustering, population structure, and PCoA analysis
Genetic diversity indexes of geographic groups and their diversity indexes
Data availability statement
Discussion
EST-SSR profiles in the transcriptome
Detection and validation of EST-SSR markers
Genetic diversity and population structure of Phalaris accessions
Conclusions
Acknowledgments
Conflict of interest
Rights and permissions
References
About this article
Next Previous
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

    Reed canary grass (Phalaris arundinacea L.) is a perennial cool-season grass with diploidy, tetraploid and hexaploid forms native to Europe, Asia, and North America[1]. As a widely distributed species, reed canary grass is adaptable to diverse environmental conditions and can grow in different habitats between 75 and 3,200 m in altitude[2]. In addition, reed canary grass has a variety of applications, Firstly, due to its short reproductive period, high tillering capacity, high yield and high regeneration capacity, reed canary grass is often used as forage, hay, or silage[3]. Secondly, reed canary grass can also be used as a bioenergy source due to the early harvesting period and the high yield of the grass, which ensures a constant supply of raw material for bioreactors and power plants[4]. Finally, reed canary grass also has the advantages of water and soil conservation, remediation of heavy metal pollution in the environment and soil improvement due to its enormous roots and thick rhizome[5−7]. However, despite its many advantages, current research on the genus Phalaris is focused on biological characteristics and forage quality, and research on cultivation and variety selection has lagged in comparison to other forage grasses[8].

    DNA markers, such as Amplified fragment length polymorphisms (AFLPs)[9], Random amplified polymorphic DNA (RAPD)[10], Single primer amplification reaction (SRAP)[11], Simple sequence repeat (SSR)[12], and Single nucleotide polymorphism (SNPs)[13], are practical tools for quantitative trait locus (QTL) mapping[14], marker-assisted selection (MAS)[15], evolutionary research, and genetic diversity analysis[16]. Especially, SSR (Simple sequence repeat) is popular for its polymorphism, abundance, codominance, sufficient variation, and cost-effectiveness[12]. SSR can be divided into genomic SSR (G-SSR) and expressed sequence tag SSR (EST-SSR)[17]. Among these, EST-SSR exhibited great application potential owing to its easy availability, good interspecies transferability, and its linkage with some traits or resistance-associated functional genes. In recent years, many EST-SSR markers have been developed in several plant species, which have high transferability in their related species, such as Thujopsis spp[18], Pseudotaxus chienii[19], and Amentotaxus spp[20]. These species' genetic diversity, genetic divergence patterns, and population genetic structure were studied using the developed markers[21]. However, few studies have reported the development of EST-SSR of reed canary grass.

    Next-generation sequencing (NGS) has become more prevalent in de novo transcriptome analysis because of technological advancements in sequencing[22]. NGS, an efficient method, is renowned for its high throughput and lower cost characteristics. Therefore, it is often used to explore expressed sequence data of non-model species[23]. Transcriptome sequencing also offered a simple and effective way for developing molecular markers, especially for heterozygous polyploidy species with a large genome. Thus, NGS technology has contributed to ecology, evolution, and conservation genetics by obtaining large quantities of accessible genomic and transcriptomic data for Gramineae species[24].

    In recent years, an increasing number of EST datasets have become available for both type and non-type plants, however, few EST-SSRs are currently available for reed canary grass. In this study, the reed canary grass transcriptome was obtained and functionally annotated to better understand its functional classification. Secondly, we have analyzed the frequency, distribution and function of SSRs of reed canary grass in the transcriptome. Finally, the genetic diversity and structure of 17 reed canary grass and two bulbous canary grass were studied using EST-SSR markers.

    Materials and methods

    Plant germplasms, RNA extraction, and DNA extraction

    The fresh leaves, roots, and stems of P. arundinacea CV. Chuanxi (tetraploid) were collected from a nursery of the Sichuan Academy of Grassland Sciences in Dayi County (32°48" N, 102°33" E), Sichuan, China. These tissues were mixed for RNA extraction, after RNA quality inspection, transcriptome sequencing was performed with three replicates. The other 18 accessions were obtained from National Plant Germplasm System (NPGS) and maintained in the growth chamber at the Sichuan Academy of Grassland Sciences. The mixed leaves of all 19 accessions were dried with silica gel until use. Total RNA was extracted using an RNA extraction kit (Tiangen Biotech, Beijing, China), and total DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method from 19 accessions. The concentration and quality of the extracted DNA were analyzed using the NanoDrop1 ND-1000 Spectrophotometer (NanoDrop Technologies, USA) and agarose gel electrophoresis, respectively[25].

    cDNA preparation and Illumina sequencing

    To construct the cDNA library, we used the SMARTTM cDNA library construction kit (Clontech, Mountain View, CA, USA). The cDNA library was constructed based on a previously described method[26], and then sequenced using Illumina HiSeq™4000 platform (2 bp × 150 bp read length) (San Diego, CA, USA) at Wuhan Genomics Institute (Frasergen, Wuhan, China).

    Transcriptome assembly and annotation

    The raw reads were filtered using the SOAPnuke v2.1.0 software. The following filtering parameters were set: discard paired reads containing splice sequences with ambiguous bases N > 5% and remove low-quality paired reads with more than 50% of the entire read length in bases with Qphred ≤ 20 (Q20). Trinity software was used to assemble transcript sequences. Finally, all transcripts are compared in a public protein database (KOG, GO, KEGG, NR, Swiss-Prot) via BLASTX. BLAST2GO (https://www.blast2go.com/) with NR annotation were used to obtain the assembled transcripts for GO annotation (Gene Ontology, GO), and metabolic pathway analysis of the assembled transcripts were performed according to the KEGG (http://www.genome.jp/kegg/) database[27−28].

    SSRs identification and primer design

    MicroSAtellite software (MISA) was used to identify SSRs within transcript sequences longer than 500 bp[29]. These SSR loci can be identified using the repeat number of mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs greater than or equal to 10, 6, 5, 5, 5, and 5, respectively. The primers were designed using Primer 3.0[30], and the principles are as follows: (1) Primer length between 18 and 25 bp; (2) An annealing temperature of 57 °C to 63 °C is recommended, with 60 °C being the optimal temperature.; (3) GC content of 30%−70%, optimal GC content of 50%; (4) amplification product length of 100−300 bp.

    EST-SSR amplification

    Three hundred EST-SSR primer pairs were randomly selected to identify polymorphism based on four geographically distant accessions. PCR amplification was performed in a volume of 20 µL; PCR reactions included 4 µL (20 ng/µL) DNA samples, forward and reverse primers, 0.5 µL each (10 mM), 0.5µL Taq enzyme (2.5 U/µL), 10 µL 2× Master Mix (Tiangen, Beijing), and 4.5 µL ddH2O. The cycling conditions were conducted as follows: initiation at 95 °C for 2 min, followed by 30 cycles of 30 s intervals at 95 °C, annealing at 45 °C for 30 s, 1 min at 72 °C, and 2 min at 72 °C. Each primer was amplified twice to determine if it produced clear and reproducible bands. To assist in detecting polymorphic bands, we electrophoresed 8% non-denaturing polyacrylamide gels with 1% TBE buffer solution with silver nitrate staining. Finally, 19 accessions were genotyped via EST-SSRs with high transferability, polymorphism, and repeatability.

    Genotyping and data analysis

    SSR is a co-dominant marker, but amplifying alleles in reed canary grass can be challenging due to its diploid, tetraploid, and hexaploid characteristics. Therefore, the amplified SSR bands are recorded as either present (1) or absent (0). Based on the objective results, only well-resolved, unambiguous bands (> 50 bp) were detected. The number of polymorphic bands (NPB) was recorded with a threshold of 5%. The polymorphic information content (PIC) was calculated using PIC = 1 − p2 − q2, it ranged from 0−0.5 and a larger PIC value indicated better polymorphism of the dominant marker, where p and q are the frequencies of present and absent, respectively[31]. The marker index (MI) was calculated using MI = PIC × NPB [32] . Resolving power (RP) was used to distinguish between genotypes in germplasm panels, which was calculated using Rp = Σ Ib. Ib was calculated using Ib = 1 − (2 × |0.5 − Pi|), where Pi is the frequency of amplification bands[32].

    GenAlex 6.51 was used to calculate the allele number (Na), the effective number of alleles (Ne), the Shannon information index (I), the expected heterozygosity (He), and pairwise population PhiPT values (Fst) among the geographical groups. PCoA was also performed with the GenAlex 6.51 program[33]. At the germplasm level, the genetic similarity coefficient (Dice) was evaluated, and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was conducted using the FREETREE software[34]. Based on bootstrap values (1,000 substitutions), Fig Tree V 1.4.3 was used to test the robustness of dendrograms[35]. The population structure was acquired using STRUCTURE software, and the optimal K value was determined using the CLUMMPP software[36−37].

    Results

    Sequencing and de novo assembly

    After rigorous quality control and data filtering, 24,836,493 high-quality mean clean reads were obtained, and 272,328 transcripts were generated using the Trinity program (China National GenBank Data Base: CNX0602781). The clean reads contain over 97.93% of sequencing bases with quality scores at the Q20 level (an error probability of 0.1%) and over 90.08% at Q30. The mean GC content of generated sequence was 54.17% (Table 1). Based on these results, the sequencing data is of sufficient quality for further analysis. As shown in Fig. 1a and Supplemental Table S1, most transcripts were < 2,000 bp in length (215,33, 79.08%). There was a general decrease in transcript number with increasing transcript length, and most transcripts were approximately 500 bp in length (81,141, 29.08%), indicating that the combination exhibited high sequencing quality (Supplemental Tables S1 & S2).

    Table 1.  De novo transcriptome sequencing of reed canary grass.
    ReplicatesReadCleanSize of cleanQ20 (%)Q30 (%)GC (%)
    LengthReads pairsBase (bp)
    Sample115024,378,7137,313,613,9009789.4553.8
    Sample215022,716,8536,815,055,90097.5591.153.7
    Sample315027,431,9128,229,573,60097.0589.755.1
    Mean15024,842,4937,452,747,80097.290.0854.2
     | Show Table
    DownLoad: CSV
    Figure 1.  Characteristics of reed canary grass transcripts. (a) Distribution of transcripts lengths in Phalaris. (b) Functional annotation of transcripts based on Gene Ontology (GO) categorization. (c) Top 19 KEGG pathways containing the most transcripts. (d) Distribution of six SSR repeat types in different genic regions.
    DownLoad: Full-Size Img PowerPoint

    Annotation and classification of Phalaris transcripts

    Four databases, NR, Swiss-Prot Annotation, GO and KEGG, were used to perform the annotation using the BLASTX algorithm with an e-value of 1.0 × 10−5. A total of 272,328 transcripts were annotated in at least one of the aforementioned databases. In the NR database, 158,464 transcripts revealed a significant number of hits (e-value < 1× 10−5) of which 8,917 were related to Artibeus jamaicensis (Supplemental Table S3) The GO database, Swiss-Prot annotation and KEGG databases successfully annotated 110,631, 106,768 and 59,324 transcripts with known proteins. However, 113,242 (41.58%) transcripts did not match any sequence in the aforementioned four databases (Table 2).

    Table 2.  Annotation statistics of reed canary grass transcripts.
    DatabaseNumber of transcriptsPercentage
    Total272,328100%
    KOG46,69717.15%
    KEGG59,32421.78%
    NR158,46458.19%
    GO110,63140.62%
    Swiss-Prot106,76839.21%
    Unknown113,2441.58%
     | Show Table
    DownLoad: CSV

    The GO annotation results revealed that the major subcategories of the classified transcripts were 'metabolic processes' (60,037), 'cellular processes' (57,599), and 'single biological processes' (35,290) in 'biological processes'; 'cells' (60,005), 'cellular fractions' (60,005) and 'organelles' (16,975) in 'cellular components'; 'catalytic activity' (57,745) in 'molecular functions'; and 'binding' (186), and 'transporter activity' (7,157) (Fig. 1b). In the KEGG pathway, the most abundant pathways were 'transport and catabolism' (2,581), 'carbon metabolism' (5,035), 'biosynthesis of amino acids' (3,429), 'signal transduction' (2,313), 'transport and catabolism' (2,581), 'folding, sorting and degradation' (4,058), and 'environment adaptation' (1,701) (Fig. 1c).

    Prediction of repeat motif types

    A total of 50,155 potential SSRs were identified from 272,328 transcripts, with 1,936 sequences containing more than one SSR locus. Of the 50,155 SSRs, 1,936 were compound microsatellites (Table 3). The type and distribution of 50,155 potential SSRs were investigated. The most abundant repeat motif was mono-nucleotide SSRs (22,859, Fig. 1d), with the vast majority (45.58%) comprising A or T repeats, followed by Tri-type (34.42%) and Di-type (17.35%). AG/CT and CCG/CGG exhibited the highest proportion of the Di-motif and Tri-type occurrences (Fig. 2, Supplemental Table S4).

    Table 3.  Statistics of SSRs identified in reed canary grass transcripts.
    SSR miningNumber
    Total number of sequences examined272,328
    Total size of examined sequences (bp)351,691,355
    Total number of identified SSRs50,155
    Number of SSR containing sequences41,925
    Number of sequences containing more than 1 SSR6,779
    Number of SSRs present in compound formation1,936
    Distribution of SSRs in different repeat types
    Mono-nucleotide22,859(45.58%)
    Di-nucleotide8,702(17.35%)
    Tri-nucleotide17,261(34.42%)
    Tetra-nucleotide824(1.64%)
    Penta-nucleotide318(0.63%)
    Hexa-nucleotide191(0.38%)
     | Show Table
    DownLoad: CSV
    Figure 2.  Simple sequence repeats length distribution across different motif classification in reed canary grass.
    DownLoad: Full-Size Img PowerPoint

    Development and polymorphism identification of EST-SSR markers

    Based on the predicted SSR markers, 300 EST-SSR primers were randomly selected and used for PCR amplification and polymorphism assessment (Supplemental Table S5). The amplification results revealed that 45 polymorphic markers (16.3%) were used to amplify the 19 reed canary grass accessions (Supplemental Tables S6 & S7). The transcripts for 45 markers were annotated, and major GO terms included 'integral component of membrane' and 'membrane' in 'Molecular Function'; 'ATP binding' in 'Biological Process' (Supplemental Fig. S1a). The KEGG annotation results revealed that the major KEGG subclass included 'Metabolism of cofactors and vitamin' and 'Biosynthesis of other secondary metabolites' in 'Metabolism' (Supplemental Fig. S1b). Supplemental Fig. S2 depicts the gel images of SSR1-SSR5. Forty-five SSR markers amplified 218 bands (TNB), and reliable polymorphic bands (NPB) amplified 216 bands (99.08%), which were amplified by each marker ranging from two (SSR17, SSR19, and SSR25) to 16 (SSR2) (Table 4). The percentage of polymorphic bands (PPB) of each marker ranged from 80% (SSR15) to 100% (SSR2, SSR3 and so on) in Table 4. Furthermore, the PIC (ranged from 0.37 to 0.43), MI (ranged from 0.75 to 4.12), Rp (ranged from 0.42 to 9.05), H (ranged from 0.38 to 0.500), and I (ranged from 0.49 to 0.72) of these 45 EST-SSR markers were high, suggesting that these markers have great application potential for the genetic study of Phalaris species (Table 4).

    Table 4.  Marker parameters calculated for each SSR primer combination used with reed canary grass accessions.
    TNBNPBPPB%PICMIRpHI
    SSR1109900.393.475.790.470.59
    SSR216161000.386.019.050.490.62
    SSR3991000.383.454.420.480.60
    SSR4661000.392.312.210.470.49
    SSR5881000.372.994.320.500.62
    SSR6771000.372.624.000.500.61
    SSR7661000.392.363.370.460.59
    SSR8771000.392.703.260.470.59
    SSR910101000.383.833.890.480.59
    SSR10771000.382.633.680.490.63
    SSR1111111000.374.126.110.500.61
    SSR12771000.382.644.210.490.67
    SSR13771000.412.863.370.420.66
    SSR14991000.383.454.320.480.6
    SSR1554800.391.562.630.470.57
    SSR16661000.372.241.050.500.56
    SSR17221000.370.750.420.500.48
    SSR18551000.412.071.160.410.63
    SSR19221000.400.791.050.450.61
    SSR20331000.391.171.260.470.59
    SSR21331000.401.211.160.430.52
    SSR22331000.371.121.370.500.62
    SSR23551000.391.972.740.450.6
    SSR24331000.391.182.110.460.72
    SSR25221000.380.771.050.480.64
    SSR26221000.380.760.950.490.61
    SSR27331000.391.180.740.460.53
    SSR28221000.380.761.580.490.66
    SSR29221000.400.791.050.450.61
    SSR30441000.391.572.110.460.56
    SSR31331000.371.122.530.500.66
    SSR32221000.410.831.160.410.72
    SSR33331000.381.132.840.490.7
    SSR34221000.400.791.050.450.56
    SSR35551000.391.953.580.470.64
    SSR36331000.381.141.580.490.62
    SSR37221000.370.751.890.500.7
    SSR38221000.380.751.050.490.64
    SSR39661000.382.304.630.480.72
    SSR40551000.432.131.790.380.52
    SSR41441000.391.572.420.460.6
    SSR42221000.380.761.580.490.71
    SSR43221000.400.790.840.450.5
    SSR44221000.380.761.680.490.67
    SSR45331000.391.161.580.470.6
    Total21821699.080.3780.74114.630.500.61
    Mean4.844.8099.330.391.854.980.470.61
    MI, marker Index; Rp, resolving power; I, Shannon information index; H, heterozygosity.
     | Show Table
    DownLoad: CSV

    Clustering, population structure, and PCoA analysis

    Genetic similarities among the tested accessions were calculated, and an unrooted UPGMA dendrogram was created. Nineteen germplasm were divided into three clusters based on their average genetic similarity values (0.9207) (Cluster I, Cluster II, and Cluster III; Fig. 3; Fig. 4). Overall, the clustering results were correlated with geographic origin, with Cluster I including six accessions from North America (NoA), four accessions from Europe (EU), and two accessions from Asia (AS); Cluster II consisting of five accessions from NoA; Cluster III consisting of bulbous canary grass (P. aquatica). (Fig. 3; Fig. 4). Structural software was used to assess the genetic membership of the studied accessions based on Bayesian models (Fig. 3; Supplemental Fig. S3). According to Evanno's method, the optimal K value was three (Supplemental Fig. S3).

    Figure 3.  Unweighted Pair Group Method with Arithmetic (UPGMA) tree of the 19 accessions (the reliability of the clustering results is indicated by a bootstrap support value of more than 50% for each main branch of the clustering tree map) and genetic relationship among reed canary grass accessions using a Bayesian analysis.
    DownLoad: Full-Size Img PowerPoint
    Figure 4.  Principal coordinate analysis (PCoA) showing the relationships of the reed canary grass accessions.
    DownLoad: Full-Size Img PowerPoint

    Genetic diversity indexes of geographic groups and their diversity indexes

    Based on the geographical origin of all germplasms, all 19 accessions were divided into four geographical groups: NoA, EU, AS, and Pa, with NoA consisting of 11 reed canary grass accessions from North America, EU consisting of four from Europe, AS consisting of two from Asia, and Pa consisting of two bulbous canary grass accessions. NoA exhibited the highest level of genetic diversity (Na = 1.955, Ne = 1.577, I = 0.512, He = 0.341, P = 96.53%; Table 5), followed by the EU, AS, and Pa groups (Table 5 & Supplemental Table S6). AMOVA is usually used to test the effect of geographic origin on the genetic variation of different germplasm. Among the total genetic variation, 2% was attributed to variation among geographic populations, while 98% was due to variation among germplasm within populations (p < 0.05; Table 6; Supplemental Fig. S4). The mean fixation index (Fst) of the three groups revealed a moderate genetic differentiation (Fst = 0.023; Table 6).

    Table 5.  Different genetic diversity estimates for four geographical groups of reed canary grass accessions.
    Geographical groupNNaNeIHeP
    NoA11.0001.9551.5770.5120.34196.53%
    EU4.0001.4951.4320.3580.24462.38%
    AS2.0000.8661.1680.1440.09823.76%
    Pa2.0000.8911.1750.1500.10324.75%
    N, Individual number of populations; Na, No. of different Alleles; Ne, No. of effective alleles; I, Shannon information index; He, Expected heterozygosity; P, Genetic variation.
     | Show Table
    DownLoad: CSV
    Table 6.  Analysis of molecular variance (AMOVA) among and within geographical groups of reed canary grass accessions.
    Source of variationdfSSMSEst. Var.PMV (%)FstP
    Among pops24.4102.2050.0462%0.0230.143
    Within pops1428.0402.0032.00398%
    Total1632.4502.049100%
    df, degree of freedom; SS, square deviation; MS, mean square deviation; Est.Var, exist variance; Fst, coefficient of genetic differentiation; PMV, Percentages of molecular variance.
     | Show Table
    DownLoad: CSV

    Data availability statement

    The Illumina NGS reads generated in this study were submitted to China National GenBank Data Base (Accession No. CNX0602781).

    Discussion

    Reed canary grass was promoted extensively as a high-yielding forage species on the northwest Sichuan plateau (China). It has superior flooding tolerance compared with other grass species, making it one of the most important grass species suitable for wetland restoration. Several germplasms of reed canary grass have been discovered on the western Sichuan plateau, resulting in cultivated or wild domesticated varieties[38]. However, because of a lack of genomic information, there are few reports on the development of molecular markers, which is unfavorable to the assisted breeding process[39]. In the present study, polymorphic EST-SSR markers were developed via the transcriptome sequencing of reed canary grass; these markers are crucial for the future genetic improvement of this ecologically and economically important plant. The identified transcripts and annotated pathways facilitate further research into the genetics of Phalaris species.

    EST-SSR profiles in the transcriptome

    EST-SSR is essential in investigating species' genetic diversity and molecular breeding[24]. EST-SSRs are closely connected to functional genes compared with G-SSRs, and EST-SSRs which usually have fewer alleles and higher transferability. In genetic diversity studies of E. excelsus, EST-SSRs have a higher generalizability (30.61%) than G-SSRs (17.86%)[40]. Based on the transcriptome sequencing of reed canary grass, we predicted an abundance of SSR loci (50,155 SSRs), and the frequency of SSR (18.42%) is much higher than that obtained from E. sibiricuss (8.19%, 1/6.95 kb)[22] and Leymus chinensi (4.38%, 1/10.78 kb)[41]. The A/T and CCG/CGG enrichment tendencies of single and trinucleotide motifs are consistent with those of eukaryotes[42]. The most abundant dinucleotide repeat motif was AG/CT (72.90%), which is also consistent with the results of Lolium multiflorum[41].

    Detection and validation of EST-SSR markers

    The aforementioned EST-SSR markers were used to study the genetic diversity of 19 reed canary grass accessions. Therefore, the present study is the first to develop SSR markers and identify and differentiate 19 accessions in various geographical regions. In this study, 45 polymorphic EST-SSR markers were identified with a higher percentage of polymorphic bands (an NPB mean of 62.15%) than most grass species, such as Elymus excelsus[10] and Bromus japonicus[43]. PIC, which is an essential index for distinguishing dominant markers, theoretically ranges from 0 to 0.5[31]. In this study, the mean PIC of the 45 SSR markers was 0.364. MI and Rp were correlated with primer identification ability. Furthermore, the mean values of MI and Rp were 0.951 and 0.956, respectively. These findings indicate that the developed markers have the potential to elaborate on the genetic diversity of Phalaris species. Among the 45 EST-SSR markers, SSR12 (PIC = 0.405, MI = 1.216, Rp = 1.143), SSR39 (PIC = 0.469, MI = 1.407, Rp = 1.211), and SSR42 (PIC = 0.465, MI = 0.931, Rp = 1.158), which exhibited high PIC, MI, and Rp values—served as optimal SSR primers for germplasm identification of reed canary grass.

    Genetic diversity and population structure of Phalaris accessions

    Cluster analysis and genetic structure are essential to studying germplasm genetic relationships[44]. Nineteen accessions were identified using UPGMA and PCOA as Cluster I, Cluster II, and Cluster III. The genetic structure patterns of the three clusters were also different from each other, which roughly correspond to their geographical sources. However, Cluster I comprised six accessions from NOA, four from EU, and two from AS. The findings suggest that geographical isolation does not necessarily lead to substantial genetic differentiation. By contrast, convergent evolution because of similar habitat conditions may account for the greater genetic similarity between geographically distant accessions[45]. It is also possible that these few abnormally clustered germplasms were historically introduced elsewhere. In the present study, two bulbous canary grass were identified as Cluster III, demonstrating that 45 newly developed SSR markers in other Phalaris species are reliable and have broad application value. Meanwhile, population structure was analyzed using structural software. The optimal K value for the analysis was three and revealed three genetic backgrounds because genetic drift, mutations, gene flow, and natural selection have weakened the structural program[46]. The genetic diversity analysis revealed that NOA (He = 0.341) had higher genetic diversity than EU (He = 0.244), AA (He = 0.274), and Pa (He = 0.103). The AMOVA analysis revealed a moderate genetic variation (Fst = 0.023, p < 0.05) between the three geographic groups, which can be attributed to two factors: firstly, the self-pollinating characteristics of the reed canary grass[47], and secondly, EST-SSRs are derived from transcripts that, despite their excellent transferability, are relatively conserved among different materials, so this conservation is due to the essential life functions for which the transcripts of the EST-SSR sources screened are responsible, including the survival and reproduction of the species[43].

    Conclusions

    In this study, transcriptome sequencing of reed canary grass was performed, and the transcripts were de novo assembled. A total of 272,328 non-redundant transcripts containing SSRs were annotated in several databases, which were associated with several biological processes. A total of 50,155 EST-SSR were identified from the assembled transcripts, and 300 EST-SSR markers were randomly selected for validation. Therefore, 45 SSR markers demonstrated high polymorphism, stable amplification, easy identification of amplified bands, and stability between accessions, thereby filling a gap in the development of SSR primers based on the transcriptome of reed canary grass.

    Acknowledgments

    This research was funded by Supported by the Sichuan Science and Technology Program, grant number 2022YFN0035; Sichuan Beef Innovation Team Project, grant number sccxtd-2019-13; Sichuan Forage Innovation Team Project, grant number sccxtd-2020-16; Sichuan Forestry and Grassland Science and Technology Innovation Team Special Funding of China, grant number LCTD2023CZ01; Sichuan Province '14th Five-Year Plan' Forage Breeding Research Project of China, grant number 2021YFYZ0013-2, and National Forage Industry Technology System Aba Comprehensive Experimental Station of China, grant number CARS-34.

  • 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
    shu

Figures(4)  /  Tables(2)

Download PDF

Article Metrics

Article views(3573) PDF downloads(539)

Other Articles By Authors

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

  • 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.’

    HTML

    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.

      Rights and permissions
      • 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)
  • About this article
    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
    shu

Catalog

  • HTML

  • Introduction
  • Materials and methods

  • Toxin preparation and assay

  • Phytotoxicity testing in different tissues of banana

  • Data collection and statistical analyses

  • Results
  • Discussion
  • Conclusions
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
  • About this article
logo

Contents

Policies

Services

Partnerships

© 2025 Maximum Academic Press, except Open Access articles. MAP is a member of ALPSP, COPE, CrossRef, cOAlition S, OASPA and STM.

Privacy Policy  |  Site Map  |  Terms and Conditions

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return