Skip to main content
Download PDF

In vitro RNA-mediated gene silencing of Fusarium oxysporum f.sp. cubense from Ecuador and assessment of RNAi molecule stability in banana plants

BMC Research Notes volume 18, Article number: 185 (2025) Cite this article

Abstract

Objective

Fusarium wilt poses a significant threat to banana production, caused by diverse clonal Fusarium lineages. Given the lack of curative measures, developing effective treatments is crucial. RNA interference (RNAi) technology, utilizing double-stranded RNA (dsRNA) molecules, offers a promising solution. In this study, RNAi was evaluated by silencing the activity of the Beta-tubulin (Focβ-tub), C5 Sterol desaturase (FocERG3) and Chitin synthase 1 (FocChs1) genes in a pathogenic Fusarium strain. Furthermore, we study the potential of dsRNA translocation in 3 months old banana plants at early hours of been spray under greenhouse conditions.

Results

In vitro results demonstrated that dsRNA-FocChs1 was more effective in inhibiting spores, with an average IC50 of 156.84 mg/L, compared to dsRNA-Focβ-tub (IC50: 532.7 mg/L), dsRNA-FocERG3 (IC50: 635.59 mg/L), and a positive control (IC50: 243.91 mg/L). A greenhouse test was conducted to evaluate the translocation of dsRNA in banana plants. The results demonstrated that the dsRNA remained on the applied leaf without degradation up to 48 h post-application (hpa). However, no translocation to other plant tissues was detected until the last time point. Further time points should be evaluated to ascertain the dsRNA translocation to other banana plant tissue.

Peer Review reports

Introduction

Bananas are one of the most important fruits worldwide, with a high demand for consumption [1, 2]. Ecuador is the prime exporter, with a volume of 6.2 million tons in 2023 [1]. Banana industry is facing a significant threat from Fusarium oxysporum f. sp. cubense (Foc), a fungus that causes Fusarium wilt of banana [2, 3]. All commercial banana varieties are currently threatened by a new Foc genetic variant, commonly referred to as Fusarium tropical race 4 (FocTR4) [4,5,6]. Foc leads to plant death by blocking the vascular bundles [7,8,9]. Currently, most fungicides are ineffective in controlling Foc [10,11,12]. Nonetheless, a new compound may be able to control Fusarium species [13]. In Ecuador, there are no reports of the presence of FocTR4 [14], but has been detected in neighboring countries [15,16,17]. RNAi is a biological process that silences virulence-inducing genes in pathogens [18]. The RNAi process involves the formation of the RNA-induced silencing complex (RISC), which degrades mRNA or suppresses target gene expression [19, 20]. RNAi-mediated host-induced gene silencing (HIGS) has been used to develop disease-resistant transgenic bananas [21]. But legal restrictions in Ecuador prohibit their cultivation [22]. Alternative, Spray-induced gene silencing (SIGS) offers an efficient, sustainable method for diseases control, reducing agrochemicals use and preventing resistance [23]. The RNAi method has been proven effective in inhibiting the growth of banana pathogenic fungi [24,25,26]. This study was conducted on the Foc race 1 strain (FocR1) from Ecuador. Notably, there is a high similarity in the sequence of target genes used between FocR1 and Foc TR4 (Additional file 3).

Main text

Materials and methods

Inoculate obtention

Assays was performed using FocR1 isolate “EC35-G-GM1” (obtained from CIBE-ESPOL collection, Ecuador) [14]. The fungus was grown and maintained at 28° C in PDA culture medium (Difco, US). To obtain spores, three 0.5 mm diameter discs were punched from the strains and transferred to 50 ml of sterile PDB medium (Difco, US) and kept for 6 days at 28° C in a shaker incubator (Innova 40, New Brunswick, Germany) at 150 rpm. The spore suspension was filtered to remove the mycelial structure and an aliquot was used to estimate the concentration using a Neubauer chamber (1/10 mm deep, Boeco, Germany), under a microscope (ZIESS, Axioskop 2 plus, Germany) at 10 X magnification.

Target genes selection

The target genes selected for RNAi were beta tubulin (Focβ-tub), C5-esterol desaturase (FocERG3) and chitin synthase 1 (FocChs1). These genes were selected according to their importance in cell function as described in Additional file 4. Furthermore, adenylate cyclase sequence from Mumbanza et al. (2013) was included as a positive control target [26]. The complete coding DNA sequence (CDS) of each target gene was obtained from “Ensembl fungi” online database [27]. Primers were designed using “Primer 3” software [28]. The T7 RNA polymerase promoter sequences (underlined in each primer in Table 1) were added to primers to enable double-stranded RNA synthesis.

Table 1 Primers F.r obtaining double-stranded RNA F.r Fusarium oxysporum F. Sp. Cubense race 1 and RT-qPCR reaction
Full size table

Synthesis of DsRNA

Total RNA was extracted from the mycelium of FocR1 using Trizol® method (Cat. 15596-018, Invitrogen, USA). Next, complementary DNA (cDNA) was obtained with the “Maxima First Strand cDNA Synthesis kit with dsDNAse” (Ref. K1642, Thermo Scientific, USA). Subsequently, RT-PCR was performed with all the target genes as described in Additional file 1. A total of 5 µl of each RT-PCR reaction was loaded for analysis via electrophoresis using a 1.5% agarose gel. The gel was run for 30 min at 10 V/cm and visualized using the GelDoc XR+ (BioRAD, USA) (Additional file 5). The dsRNA synthesis for inhibition and translocation assays was carried out following the instructions provided in the “MEGAscript™ T7 Kit” (Ref. AM1334, Invitrogen, USA). 5 µl of each RT-PCR product was used as a template, with a final reaction volume of 20 µl. dsRNA was quantified using the Nanodrop 2000 (Invitrogen, USA).

FocR1 DsRNA spore Inhibition bioassays

To study the antifungal effects of dsRNA-FocChs1, dsRNAFocβ-tub, dsRNAFocERG3 and dsRNAFocAdcy on fungal growth, assays were performed to quantify the reduction in the number of FocR1 colonies. The methodology described by Bailey et al. (2010), Mumbanza et al. (2013) [26, 30] were followed as described in Additional file 1. The dsRNA was tested at concentrations of 100, 250, 500, 1000 and 1500 mg/L with nine replicates for each target gene. Data from inhibition and survival percentages were used to independently calculate the IC50 for nine replicates, which were then plotted and fitted to the IC50%.FUN model using Kyplot 6.0 software (KyensLab Inc., Tokyo, Japan) (Additional file 1) [31, 32]. A gene expression assay was conducted with FocChs1, examining the interaction between FocR1 and dsRNA-FocChs1 in PDB medium for 24 and 48 h of Foc grow (Additional file 1).

DsRNA translocation bioassay in banana plants

A greenhouse assay was conducted to study the translocation of the dsRNA in banana plants. The dsRNA-FocAdcy was synthesized using the MEGAscript™ T7 Kit (Ref. AM1334, Invitrogen, USA). The solution, comprising 500 mg/L dsRNA-FocAdcy, 1% Triton X (Sigma, USA) and 1% DMSO (Sigma, USA), was sprayed on the upper and lower surfaces of leaf number 3 of 3-month-old banana plants of the Williams variety (Genotype AAA), which were approximately 30 cm in height. Two biological replicates (due to limited dsRNA solution) were used. Samples were collected at 1-, 12-, 24-, and 48-hours post-application (hpa) from pseudostem, corm, root, and leaf number 2, 3, and 4. Upon collection, the samples were immediately frozen with liquid nitrogen and stored at -80 °C until processing. For total RNA extraction, frozen samples were pulverized in liquid nitrogen using a MM400 mixer-mill (Retsh, Hann, Germany). From each ground tissue, 100 mg was used for total RNA extraction with the “Spectrum™ Plant Total RNA Kit” (Ref. STRN50-1KT, Sigma, USA). Total RNA was quantified using the Nanodrop 2000 (Invitrogen, USA).

Northern blot detection of DsRNA in different tissues of banana plants

The Northern blot technique was used to detect the mobility of the dsRNA using total RNA from all tissue samples. This highly sensitive method was developed following the manufacturer’s instructions for the “Dig Northern Starter Kit” (Cat. No 12039672910, Roche Switzerland). Probes generated are described in Additional file 2 [33,34,35]. To detect luminescence, immunological detection of RNA via probe hybridization on a membrane was performed using the Stella 3200 (Raytest, Germany) with 20 min of exposure in dark conditions. The resulting image was analyzed with the “AIDA Image Analyzer” software (Raytest, Germany).

Statistical analysis

Data analysis and plotting were performed using R Studio [36]. To compare IC50 values and spore inhibition percentages, statistical analyses were conducted to an analysis of variance (ANOVA), followed by Fisher’s Least Significant Difference (LSD) test. A significance level of p < 0.05 was used to assess differences between treatment. Student’s T-test at P < 0.05 was use for the significance analysis results of RT-qPCR.

Results and discussions

Inhibition of FocR1 spores

All dsRNA were able to inhibit FocR1 growth (Additional file 7). At a concentration of 1000 mg/L dsRNA-Chs1 and dsRNA-FocAdcy achieved 100% inhibition approximately, while dsRNA-Focβ-tub and dsRNA-ERG3 showed 70.64% and 81.3% inhibition, respectively. At 1500 mg/L, there was no significant difference in inhibition for dsRNA-Focβ-tub and dsRNA-ERG3, with both showing 75.87% and 80.18% inhibition, respectively (Fig. 1a and b). Considering the role of the genes in the host organism, we analyzed the inhibition behavior of each dsRNA independently.

The FocAdcy gene was chosen as a control for inhibition based on previous research [26]. However, our results differ significantly from those described by Mumbanza et al. (2013), where, an inhibition percentage of 93% was reported with a dsRNA concentration of 0.6 mg/L, which is significantly lower than that observed in our investigation. However, in a study conducted by Fei et al. (2018), the inhibition of three target genes by RNAi was found to halt the virulence and spore growth of Fo5176 affecting Arabidopsis thaliana at higher concentrations. The study demonstrated a significant reduction in spore germination, with 118-fold, 153-fold, and 100-fold decreases for the target genes GenA, GenB and GenC, respectively, compared to the control using 2.5 µg of dsRNA in 25 µl (100 mg/L). The authors suggest these genes may offer more control than those in Mumbanza’s study, though the dsRNA concentration differed significantly between the studies [25]. It should be noted that the strains or forma specialis used in the studies are different from those we used, which may explain the variability.

Fig. 1
figure 1

Spore inhibition of Fusarium oxysporum f. sp. cubense race 1 with dsRNA. (a) Inhibition with dsRNA at 1000 mg/L on isolate EC35-G-GM1 of FocR1 with dsRNA-FocChs1, dsRNA-FocB-tub, dsRNA-FocERG3 and dsRNA-FocAdcy. (b) Bar graph of spore inhibition with dsRNA-FocChs1 (100%), dsRNA-FocB-tub (75.87%), dsRNA-FocERG3 (80.18%) and dsRNA-FocAdcy (99.37%) at concentrations of 1500 mg/L on spores of isolate EC35-G-GM1 of FocR1. Bars represent standard errors. Equal letters above bars are not significantly different at p > 0.05. (c) Box plot of the mean inhibitory concentration (IC50) in mg/L with dsRNA-FocChs1, dsRNA-FocAdcy, dsRNA-FocB-tub and dsRNA-FocERG3, on isolate EC35-G-GM1 of FocR1. 9 observations were included for each gene. (d) Expression level of FocChs1 gene following Foc spore growth with dsRNA-FocChs1 at 24 and 48 h (hrs). The expression level of FocChs1 was normalized to the expression of Foc GAPDH. Expression level was performed by RT-qPCR in three technical replicates and three biological replicates independently. Statistical significance (Student’s t-test) between the treatment and the control: *, P < 0.05

Full size image

According to the IC50 calculation of dsRNA-FocChs1, dsRNA-FocAdcy, dsRNA-Focβ-tub and dsRNA-ERG3, the mean concentrations were 156.84 mg/L, 243.91 mg/L, 536.7 mg/L and 635.59 mg/L respectively. Fisher’s mean comparison analysis revealed the presence of two statistically different homologous groups mark group A comprising dsRNA-Chs1 and dsRNA-FocAdcy and group B comprising dsRNA-Focβ-tub and dsRNA-ERG3. Furthermore, dsRNA-Chs1 had the lowest IC50 value with the strain tested, indicating a higher efficiency. dsRNA-Chs1 was slightly more efficient than the control gene dsRNA-FocAdcy (Additional file 6). In Fig. 1c, the box plot illustrates the behavior of all the observations of the IC50 calculation with respect to each dsRNA, showing a greater variability with dsRNA-Focβ-tub and dsRNA-ERG3 than with dsRNA-Chs1 and dsRNA-FocAdcy, which show a high and stable inhibition in the analysis. Additionally, the relative mRNA expression level of FocChs1 was reduced in the dsRNA-FocChs1 in vitro interaction at 24 and 48 h of Foc growth, with expression levels of 0.53 and 0.33, respectively, compared to a control (Fig. 1d).

Several genes are involved in the chitin biosynthetic process. However, a study showed that these genes lack compensatory mechanisms [37]. Therefore, the inhibitory effect observed in this study is likely due to the effective degradation of the FocChs1 mRNA. Conversely, with FocERG3, 25 other genes are related to the metabolic pathway of ergosterol production participate in a regulatory compensation mechanism utilizing alternative ergosterol production routes, suggesting that ERG3 is a non-essential gene [38,39,40]. This would explain the suboptimal efficacy of dsRNA-ERG3 silencing. In relation to Focβ-tub, at least four genes related to its function are known: α1- tubulin, α2- tubulin, β1- tubulin and β2- tubulin. The sequence used in this study is related to β2- tubulin, which colocalizes with β1- tubulin, indicating that they share a specific region. Studies on the functional roles of these genes suggest that β1- tubulin can compensate for the growth function in a ∆β2- tubulin mutant [41]. This may partly explain the partial inhibition of dsRNA-Focβ-tub observed in our assay.

Translocation of the DsRNA molecule

The results showed that dsRNA-FocAdcy was only detected in leaf number 3, corresponding to the tissue of application (Fig. 2). In contrast, the dsRNA was not observed in the other evaluated tissues during the analyzed time points. This finding suggests that the dsRNA-FocAdcy did not translocate to other plant tissues in the timeframe tested. Currently, there is limited information available on the uptake and translocation of dsRNA in banana plants. Nevertheless, studies conducted on barley, Arabidopsis thaliana, and grape have demonstrated that the RNAi can be transported through phloem to other tissues, thereby inhibiting the target pathogen that affects these crops [42,43,44]. However, Qiao et al. (2021) demonstrate using confocal microscopy laser scanner (CMLS) that dsRNA can be taken up by fungi, but the efficacy of silencing depends on the eukaryotic fungal species and cell types [45].

Fig. 2
figure 2

Detection of dsRNA by Northern blot in banana tissue samples. Samples were collected at 1, 12, 24 and 48 h post-application, h3 corresponds to leaf #3 of banana the plant. Ctrl, corresponds to the dsRNA control (500 mg/L) with 1% Triton + 1% DMSO. Samples from each time point evaluated were processed individually, except those corresponding to 24 and 48 h post-application (hpa), which were analyzed together in a single Northern blot experiment

Full size image

A possible reason for the inability to detect translocated dsRNA in banana plants assay could be the reduction in dsRNA concentration influenced by factors such as greenhouse conditions, physical barriers on the banana leaf surface and the presence of nucleases [46,47,48]. Even with the addition of a surfactant to the solution during spraying, some dsRNA may still be lost to the environment [46]. Conversely, the detection of dsRNA translocation may require a longer evaluation period, as the molecule’s quantity may be reduced in other tissues. However, our results demonstrate that the dsRNA remains stable at the application site under greenhouse conditions, at least during the first 48 h of evaluation. Northern blotting detection of the dsRNA-FocAdcy molecule in the leaves where the solution was applied showed that the dsRNA remained intact at all evaluated time points.

Limitation

dsRNA was not detected in tissues other than those to which it was applied. To improve detection, further translocation bioassays are being conducted with higher dsRNA concentrations, direct application to the banana corm and longer evaluation period.

Data availability

The authors declare that all relevant data generated is available in the Tables and Figures of the manuscript. The sequences of the target genes used in this study are found in the EmseblFungi database (Link: https://fungi.ensembl.org/index.html) under the following Ensembl ID codes and links: FOC1_g10012728 (Chs1) (link: https://fungi.ensembl.org/Multi/Search/Results?species=all;idx=;q=FOC1_g10012728;site=ensemblunit), FOC1_g10011241 (β-tub) (link: https://fungi.ensembl.org/Multi/Search/Results?species=all;idx=;q=FOC1_g10011241;site=ensemblunit), FOC1_g10010052 (ERG3 - Putative C-5 sterol desaturase) (link: https://fungi.ensembl.org/Multi/Search/Results?species=all;idx=;q=FOC1_g10010052;site=ensemblunit), FOC1_g10015438 (AdcyFocR1) (link:https://fungi.ensembl.org/Multi/Search/Results?species=all;idx=;q=FOC1_g10015438;site=ensemblunit ), and FOC4_g10015430 (AdcyFocTR4) (link: https://fungi.ensembl.org/Multi/Search/Results?species=all;idx=;q=FOC4_g10015430;site=ensemblunit). The gene expression of FocChs1 data supporting this study have been provided as Additional File 12.

Abbreviations

RNAi:

RNA interference

dsRNA:

Double stream RNA

mRNA:

Messenger RNA

IC50 :

Half maximal inhibitory concentration

References

  1. FAO. Banana market Review– Preliminary results 2023. Rome; 2023.

  2. Voora V, Larrea C, Bermudez S. Global market report: bananas. International Institute for Sustainable Development; 2020.

  3. Buddenhagen I. Understanding strain diversity in Fusarium oxysporum f. Sp. Cubense and history of introduction of «tropical race 4» to better manage banana production. En: Acta Horticulturae [Internet]. International Society for Horticultural Science (ISHS), Leuven, Belgium; 2009. pp. 193–204. Disponible en: https://doiorg.publicaciones.saludcastillayleon.es/10.17660/ActaHortic.2009.828.19

  4. Dita M, Barquero M, Heck D, Mizubuti ESG, Staver CP. Fusarium wilt of banana: current knowledge on epidemiology and research needs toward sustainable disease management. Front Plant Sci [Internet]. 19 de octubre de 2018 [citado 12 de julio de 2024];9. Disponible en: https://www.frontiersin.org/journals/plant-science/articles/https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fpls.2018.01468/full

  5. Maryani N, Lombard L, Poerba YS, Subandiyah S, Crous PW, Kema GHJ. Phylogeny and genetic diversity of the banana F.sarium wilt pathogen Fusarium oxysporum F. Sp. cubense in the Indonesian centre of origin. Stud Mycol 1 De Marzo De. 2019;92:155–94.

    Article  CAS  Google Scholar 

  6. Ploetz RC. Fusarium wilt of banana is caused by several pathogens referred to as F.sarium oxysporum F. Sp. cubense. Phytopathology® junio de. 2006;96(6):653–6.

    Article  Google Scholar 

  7. García-Bastidas F, Ordóñez N, Konkol J, Al-Qasim M, Naser Z, Abdelwali M, Salem N, Waalwijk C, Ploetz RC, Kema GHJ. First report of F.sarium oxysporum F. Sp. cubense tropical race 4 associated with Panama disease of banana outside Southeast Asia. Plant Dis 29 De Octubre De. 2013;98(5):694–694.

    Article  Google Scholar 

  8. Guo L, Han L, Yang L, Zeng H, Fan D, Zhu Y, Feng Y, Wang G, Peng C, Jiang X, Zhou D, Ni P, Liang C, Liu L, Wang J, Mao C, Fang X, Peng M, Huang J. Genome and transcriptome analysis of the F.ngal pathogen F.sarium oxysporum F. Sp. cubense causing banana vascular wilt disease. PLOS ONE 17 De Abril De. 2014;9(4):e95543.

    Article  Google Scholar 

  9. Ploetz R. Fusarium Wilt of Banana. Phytopathology®. 9 de junio de 2015;105(12):1512-21.

  10. Damodaran T, Rajan S, Muthukumar M, Ram Gopal, Yadav K, Kumar S, Ahmad I, Kumari N, Mishra VK, Jha SK. Biological Management of Banana Fusarium Wilt Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 Using Antagonistic Fungal Isolate CSR-T-3 (Trichoderma reesei). Front Microbiol [Internet]. 2020 [citado 2 de octubre de 2023];11. Disponible en: https://www.frontiersin.org/articles/https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fmicb.2020.595845

  11. Orr R, Nelson PN. Impacts of soil abiotic attributes on fusarium wilt, focusing on bananas. Appl Soil Ecol 1 De Diciembre De. 2018;132:20–33.

    Google Scholar 

  12. Siamak SB, Zheng S. Banana Fusarium Wilt (Fusarium oxysporum f. sp. cubense) Control and Resistance, in the Context of Developing Wilt-resistant Bananas Within Sustainable Production Systems. Hortic Plant J. 1 de septiembre de. 2018;4(5):208– 18.

  13. Sun H, Cai S, Deng Y, Cao S, Yang X, Lu Y, Li W, Chen H. Efficacy of Cyclobutrifluram in controlling fusarium crown rot of wheat and resistance risk of three Fusarium species to Cyclobutrifluram. Pestic Biochem Physiol. 1 de enero de 2024;198:105723.

  14. Magdama F, Monserrate-Maggi L, Serrano L, García Onofre J, Jiménez-Gasco M. Del M. Genetic diversity of F.sarium oxysporum F. Sp. cubense, the F.sarium wilt pathogen of banana, in Ecuador. Plants Septiembre De. 2020;9(9):1133.

    CAS  Google Scholar 

  15. García-Bastidas FA, Quintero-Vargas JC, Ayala-Vasquez M, Schermer T, Seidl MF, Santos-Paiva M, Noguera AM, Aguilera-Galvez C, Wittenberg A, Hofstede R, Sørensen A, Kema GHJ. First report of fusarium wilt tropical race 4 in Cavendish bananas caused by fusarium odoratissimum in Colombia. Plant Dis Marzo De. 2020;104(3):994–994.

    Article  Google Scholar 

  16. Acuña R, Rouard M, Leiva AM, Marques C, Olortegui JA, Ureta C, Cabrera-Pintado RM, Rojas JC, Lopez-Alvarez D, Cenci A, Cuellar WJ, Dita M. First report of F.sarium oxysporum F. Sp. cubense tropical race 4 causing F.sarium wilt in Cavendish bananas in Peru. Plant Dis Agosto De. 2022;106(8):2268.

    Article  Google Scholar 

  17. Mejías Herrera R, Hernández Y, Magdama F, Mostert D, Bothma S, Paredes Salgado EM, Terán D, González E, Angulo R, Angel L, Rodríguez Y, Ortega R, Viljoen A, Marys EE. First report of F.sarium wilt of Cavendish bananas caused by F.sarium oxysporum F. Sp. cubense tropical race 4 in Venezuela. Plant Dis Octubre De. 2023;107(10):3297.

    Article  Google Scholar 

  18. Hung YH, Slotkin RK. The initiation of RNA interference (RNAi) in plants. Curr Opin Plant Biol 1 De Junio De. 2021;61:102014.

    Article  CAS  Google Scholar 

  19. Borges F, Martienssen RA. The expanding world of small RNAs in plants. Nat Rev Mol Cell Biol Diciembre De. 2015;16(12):727–41.

    Article  CAS  Google Scholar 

  20. Noriega D, Valencia A, Villegas B. ARN de Interferencia (ARNi): Una tecnología novedosa Con potencial Para El Control de insectos Plaga. Rev UDCA Actual Divulg Científica. 2016;19(1):25–35.

    Google Scholar 

  21. Sankari RD, Varanavasiappan S, Arul L, Aiyanathan KEA, Kokiladevi E, Kumar KK. Transgenic technologies for fusarium wilt management in banana. Genetic engineering of crop plants for food and health security: volume 1. En: Springer; 2024. pp. 289–304.

    Google Scholar 

  22. Constitution of the Republic of Ecuador, biodiversity and natural resources. Official Registry No. 449, October 20. 2008, Biodiversity Article 401 (2008). 2008.

  23. Mezzetti B, Smagghe G, Arpaia S, Christiaens O, Dietz-Pfeilstetter A, Jones H, Kostov K, Sabbadini S, Opsahl-Sorteberg HG, Ventura V, Taning CNT, Sweet J. RNAi: what is its position in agriculture? J Pest Sci 1 De Septiembre De. 2020;93(4):1125–30.

    Article  Google Scholar 

  24. Ghag SB, Ganapathi TR. RNAi-mediated protection against banana diseases and pests. 3 Biotech. 2019;9(3):112.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Fei S, Constantin M, Peters J, Batley J, Aitken E, Mitter N. RNAi-based management for Fusarium wilt of banana. En: International Symposia on Tropical and Temperate Horticulture-ISTTH2016 1205. 2018. pp. 717– 20.

  26. Mumbanza FM, Kiggundu A, Tusiime G, Tushemereirwe WK, Niblett C, Bailey A. In vitro antifungal activity of synthetic DsRNA molecules against two pathogens of banana, F.sarium oxysporum F. Sp. cubense and Mycosphaerella F.jiensis. Pest Manag Sci. 2013;69(10):1155–62.

    Article  CAS  PubMed  Google Scholar 

  27. Martin FJ, Amode MR, Aneja A, Austine-Orimoloye O, Azov AG, Barnes I, Becker A, Bennett R, Berry A, Bhai J, Bhurji SK, Bignell A, Boddu S, Branco Lins PR, Brooks L, Ramaraju SB, Charkhchi M, Cockburn A, Da Rin Fiorretto L, Davidson C, Dodiya K, Donaldson S, El Houdaigui B, El Naboulsi T, Fatima R, Giron CG, Genez T, Ghattaoraya GS, Martinez JG, Guijarro C, Hardy M, Hollis Z, Hourlier T, Hunt T, Kay M, Kaykala V, Le T, Lemos D, Marques-Coelho D, Marugán JC, Merino GA, Mirabueno LP, Mushtaq A, Hossain SN, Ogeh DN, Sakthivel MP, Parker A, Perry M, Piližota I, Prosovetskaia I, Pérez-Silva JG, Salam AIA, Saraiva-Agostinho N, Schuilenburg H, Sheppard D, Sinha S, Sipos B, Stark W, Steed E, Sukumaran R, Sumathipala D, Suner MM, Surapaneni L, Sutinen K, Szpak M, Tricomi FF, Urbina-Gómez D, Veidenberg A, Walsh TA, Walts B, Wass E, Willhoft N, Allen J, Alvarez-Jarreta J, Chakiachvili M, Flint B, Giorgetti S, Haggerty L, Ilsley GR, Loveland JE, Moore B, Mudge JM, Tate J, Thybert D, Trevanion SJ, Winterbottom A, Frankish A, Hunt SE, Ruffier M, Cunningham F, Dyer S, Finn RD, Howe KL, Harrison PW, Yates AD, Flicek P. Ensembl 2023. Nucleic Acids Res. 6 de enero de 2023;51(D1):D933–41.

  28. Kõressaar T, Lepamets M, Kaplinski L, Raime K, Andreson R, Remm M. Primer3_masker: integrating masking of template sequence with primer design software. Bioinf 1 De Junio De. 2018;34(11):1937–8.

    Google Scholar 

  29. Chauhan S, Rajam MV. RNAi-mediated down-regulation of F.sciclin-like proteins (FoFLPs) in Fusarium oxysporum F. Sp. lycopersici results in reduced pathogenicity and virulence. Microbiol Res 1 De Julio De. 2022;260:127033.

    Article  CAS  Google Scholar 

  30. Bailey AM, Niblett C. Bioassay for gene Silencing constructs. U S Patent Application No. 2010;12/753:901.

    Google Scholar 

  31. Yoshioka K. KyPlot — A User-oriented tool for statistical data analysis and visualization. Comput Stat 1 De Septiembre De. 2002;17(3):425–37.

    Article  Google Scholar 

  32. Sebaugh JL. Guidelines for accurate EC50/IC50 Estimation. Pharm Stat. 2011;10(2):128–34.

    Article  CAS  PubMed  Google Scholar 

  33. De la Rosa C, Reyes JL. Northern Blot Analysis of microRNAs and Other Small RNAs in Plants. En: de Folter S, editor. Plant MicroRNAs: Methods and Protocols [Internet]. New York, NY: Springer; 2019 [citado 9 de noviembre de 2023]. pp. 121-9. (Methods in Molecular Biology). Disponible en: https://doiorg.publicaciones.saludcastillayleon.es/10.1007/978-1-4939-9042-9_9

  34. Wiegard JC, Schlüter MAC, Burenina OY, Kubareva EA, Klug G, Grünweller A, Hartmann RK. Northern Blot Detection of Tiny RNAs. En: Rederstorff M, editor. Small Non-Coding RNAs: Methods and Protocols [Internet]. New York, NY: Springer US; 2021 [citado 9 de noviembre de 2023]. pp. 41–58. (Methods in Molecular Biology). Disponible en: https://doiorg.publicaciones.saludcastillayleon.es/10.1007/978-1-0716-1386-3_5

  35. Martinho C, Lopez-Gomollon S. Detection of MicroRNAs by Northern Blot. En: Dalmay T, editor. MicroRNA Detection and Target Identification: Methods and Protocols [Internet]. New York, NY: Springer US; 2023 [citado 30 de octubre de 2024]. pp. 47–66. Disponible en: https://doiorg.publicaciones.saludcastillayleon.es/10.1007/978-1-0716-2982-6_4

  36. RStudio RsT. Integrated development environment for R. RStudio PBC, Boston. MA USA. 2020.

  37. Martín-Udíroz M, Madrid MP, Roncero MIG. Role of Chitin synthase genes in fusarium oxysporum. Microbiology. 2004;150(10):3175–87.

    Article  PubMed  Google Scholar 

  38. Bhattacharya S, Esquivel BD, White TC. Overexpression or deletion of ergosterol biosynthesis genes alters doubling time, response to stress agents, and drug susceptibility in Saccharomyces cerevisiae. mBio 24 De Julio De. 2018;9(4). https://doiorg.publicaciones.saludcastillayleon.es/10.1128/mbio.01291-18.

  39. Jordá T, Puig S. Regulation of ergosterol biosynthesis in Saccharomyces cerevisiae. Genes 15 De Julio De. 2020;11(7):795.

    Google Scholar 

  40. Deng GM, Yang QS, He WD, Li CY, Yang J, Zuo CW, Gao J, Sheng O, Lu SY, Zhang S, Yi GJ. Proteomic analysis of conidia germination in F.sarium oxysporum F. Sp. cubense tropical race 4 reveals new targets in ergosterol biosynthesis pathway F.r controlling F.sarium wilt of banana. Appl Microbiol Biotechnol 1 De Septiembre De. 2015;99(17):7189–207.

    Article  CAS  Google Scholar 

  41. Zhu Y, Zhang Y, Duan Y, Shi D, Hou Y, Song X, Wang J, Zhou M. Functional roles of α1-, α2-, β1-, and β2-Tubulins in vegetative growth, microtubule assembly, and sexual reproduction of fusarium graminearum. Appl Environ Microbiol 28 De Septiembre De. 2021;87(20):e00967–21.

    CAS  Google Scholar 

  42. Biedenkopf D, Will T, Knauer T, Jelonek L, Furch ACU, Busche T, Koch A. Systemic spreading of exogenous applied RNA biopesticides in the crop plant Hordeum vulgare. ExRNA 19 De Agosto De. 2020;2(1):12.

    Article  Google Scholar 

  43. Nerva L, Sandrini M, Gambino G, Chitarra W. Double-Stranded RNAs (dsRNAs) as a sustainable tool against Gray mold (Botrytis cinerea) in grapevine: effectiveness of different application methods in an Open-Air environment. Biomolecules. 2020;10(2).

  44. Numata K, Ohtani M, Yoshizumi T, Demura T, Kodama Y. Local gene Silencing in plants via synthetic DsRNA and carrier peptide. Plant Biotechnol J. 2014;12(8):1027–34.

    Article  CAS  PubMed  Google Scholar 

  45. Qiao L, Lan C, Capriotti L, Ah-Fong A, Nino Sanchez J, Hamby R, Heller J, Zhao H, Glass NL, Judelson HS, Mezzetti B, Niu D, Jin H. Spray-induced gene Silencing for disease control is dependent on the efficiency of pathogen RNA uptake. Plant Biotechnol J. 2021;19(9):1756–68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bennett M, Deikman J, Hendrix B, Iandolino A. Barriers to efficient foliar uptake of DsRNA and molecular barriers to DsRNA activity in plant cells. Front Plant Sci [Internet]. 12 de junio de 2020 [citado 29 de abril de 2024];11. Disponible en: https://www.frontiersin.org/journals/plant-science/articles/https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fpls.2020.00816/full

  47. Das PR, Sherif SM. Application of Exogenous dsRNAs-induced RNAi in Agriculture: Challenges and Triumphs. Front Plant Sci [Internet]. 2020 [citado 16 de octubre de 2023];11. Disponible en: https://www.frontiersin.org/articles/https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fpls.2020.00946

  48. Hoang BTL, Fletcher SJ, Brosnan CA, Ghodke AB, Manzie N, Mitter N. RNAi as a foliar spray: efficiency and challenges to field applications. Int J Mol Sci Enero De. 2022;23(12):6639.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. Gert Kema from Wageningen University & Research, Phytopathology department for editing, advices and funding contributions to this article.

Funding

acknowledgements.

We thank the ESPOL Polytechnic University (ESPOL), Wageningen University & Research, Phytopathology department and the Biotechnology Research Center of Ecuador (CIBE), grant CIBE-24-2021 for funding this research.

Author information

Authors and Affiliations

  1. ESPOL, Centro de Investigaciones Biotecnológicas del Ecuador, ESPOL Polytechnic University, Campus Gustavo Galindo, Km 30.5 Vía Perimetral, Guayaquil, 090902, Ecuador

    Ricardo Pacheco, Julio Bonilla, Aracely Paguay, Freddy Magdama & Pablo Chong

  2. Escuela Superior Politécnica del Litoral, ESPOL, Facultad de Ciencias de la Vida, ESPOL Polytechnic University, Campus Gustavo Galindo, Km. 30.5 vía Perimetral, P,O. Box 09-01-5863, Guayaquil, Ecuador

    Julio Bonilla, Freddy Magdama & Pablo Chong

Authors
  1. Ricardo Pacheco
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Julio Bonilla
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Aracely Paguay
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Freddy Magdama
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Pablo Chong
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

R.P. Wrote the original draft, prepared the figures, and did the experiments and data analysis. A.P. help with the in vitro experiments and data analysis. R.P., J.B., A.P., F.M. and P.Ch. review and edited the article. P.Ch. was the project administrator and find the resources and funding for the research and P.Ch. and J.B., did the conceptualization and validations of the experiments.

Corresponding author

Correspondence to Pablo Chong.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2: Description of probes generated for the Northen blot.

Supplementary Material 3: Sequence alignment of Target genes from FocR1 and FocTR4 results in NCBI database.

Supplementary Material 4: Description of target genes for silencing of Fusarium oxysporum f. sp. cubense race 1.

13104_2025_7253_MOESM5_ESM.docx

Supplementary Material 5: RT-PCR amplification of target genes from isolate EC35-G-GM1 of Fusarium oxysporum f. sp. cubense race 1.

13104_2025_7253_MOESM6_ESM.docx

Supplementary Material 6: Fisher’s least significant difference (LSD) test with the IC50 of target genes for EC35-G-GM1 inhibition of F. oxysporum f. sp. cubense race 1.

Supplementary Material 7: Inhibitory curve of dsRNA-FocChs1, dsRNA-Focβ-tub, dsRNA-FocERG3, dsRNA-FocAdcy.

Supplementary Material 8: Raw Data of Northern Blot Results.

Supplementary Material 9: Raw image of the first section of the Northern blot.

Supplementary Material 10: Raw image of the second section of the Northern blot.

Supplementary Material 11: Raw image of the third section of the Northern blot.

Supplementary Material 12: FocChs1 gene expression data.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pacheco, R., Bonilla, J., Paguay, A. et al. In vitro RNA-mediated gene silencing of Fusarium oxysporum f.sp. cubense from Ecuador and assessment of RNAi molecule stability in banana plants. BMC Res Notes 18, 185 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13104-025-07253-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13104-025-07253-9

Keywords

BMC Research Notes

ISSN: 1756-0500

Contact us