Foc TR4 Research Articles

Integrated control of Fusarium wilt in banana by Bacillus velezensis EB1 and potassium sorbate

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a widely distributed soilborne disease that poses a serious threat to banana production. Many control measures have been implemented but have not been effective. Here, we evaluated a combined strategy for Fusarium wilt control that involves a biological agent (Bacillus velezensis strain EB1) and a bioactive compound (potassium sorbate). Our results showed that potassium sorbate inhibited Foc TR4 in a dose-dependent manner. Potassium sorbate did not limit the growth of EB1 in vitro; instead, it promoted the growth and antagonistic ability of EB1 by upregulating the expression of antagonism-related genes. In greenhouse experiments, the combined application of EB1 and potassium sorbate significantly reduced the disease index of Fusarium wilt by suppressing fungal growth in the roots and promoting plant growth. Overall, our results demonstrated that potassium sorbate and B. velezensis EB1 can be used together for the sustainable management of banana Fusarium wilt.

Antimicrobial mechanisms and antifungal activity of compounds generated by banana rhizosphere Pseudomonas aeruginosa Gxun-2 against fusarium oxysporum f. sp. cubense.

Fusarium wilt of banana, also recognized as Panama disease, is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (FOC TR4). In recent years, strategies utilizing biocontrol agents, comprising antifungal microorganisms and their associated bioactive compounds from various environments, have been implemented to control this destructive disease. Our previous study showed that Pseudomonas aeruginosa Gxun-2 had significant antifungal effects against FOC TR4. However, there has been little scientific investigation of the antibacterial or antifungal activity. The aim of this study was to isolate, identify and evaluate the inhibition strength of active compounds in P. aeruginosa Gxun-2, so as to explain the mechanism of the strain inhibition on FOC TR4 from the perspective of compounds. The main antibacterial compounds of strain Gxun-2 were isolated, purified and identified using by fermentation extraction, silica gel column chromatography, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) techniques. The effect of the compounds on the mycelial growth, morphology and spore germination of strain FOC TR4 was observed by 96-well plate method and AGAR diffusion method. Among the metabolites produced by the strain, four antifungal compounds which were identified phenazine (C12H8N2), phenazine-1-carboxylic acid (PCA) (C13H8N2O2), 2-acetamidophenol (C8H9NO2) and aeruginaldehyde (C10H7NO2S) were identified through HPLC and NMR. Of these compounds, phenazine and PCA exhibited the most pronounced inhibitory effects on the spore germination and mycelial growth of FOC TR4. Phenazine demonstrated potent antifungal activity against FOC TR4 with a minimum inhibitory concentration (MIC) of 6.25 mg/L. The half-maximal effective concentration (EC50) was calculated to be 26.24 mg/L using the toxicity regression equation. PCA exhibited antifungal activity against FOC TR4 with an MIC of 25 mg/L and an EC50 of 89.63 mg/L. Furthermore, phenazine and PCA triggered substantial morphological transformations in the mycelia of FOC TR4, encompassing folding, bending, fracturing, and diminished spore formation. These findings indicate that strain Gxun-2 plays a crucial role in controlling FOC TR4 pathogenesis, predominantly through producing the antifungal compounds phenazine and PCA, and possesses potential as a cost-efficient and sustainable biocontrol agent against Fusarium wilt of banana in forthcoming times.

A ribonuclease T2 protein FocRnt2 contributes to the virulence of Fusarium oxysporum f. sp. cubense tropical race 4.

Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a major disease of banana plants worldwide. Effector proteins play critical roles in banana-Foc TR4 interaction. Our previous studies highlighted a ribonuclease protein belonging to the T2 family (named as FocRnt2) in the Foc TR4 secretome, which was predicted to be an effector. However, its biological function in Foc TR4 infection is still unclear. Herein, we observed significant expression of FocRnt2 during the early stage of fungal infection in planta. A yeast signal sequence trap assay showed that FocRnt2 contained a functional signal peptide for secretion. FocRnt2 possessed ribonuclease activity that could degrade the banana total RNA invitro. Subcellular localization showed that FocRnt2 was localized in the nucleus and cytoplasm of Nicotiana benthamiana leaves. Transient expression of FocRnt2 suppressed the expression of salicylic acid- and jasmonic acid-signalling marker genes, reactive oxygen species accumulation, and BAX-mediated cell death in N. benthamiana. FocRnt2 deletion limited fungal penetration, reduced fusaric acid biosynthesis in Foc TR4, and attenuated fungal virulence against banana plants, but had little effect on Foc TR4 growth and sensitivity to various stresses. Furthermore, FocRnt2 deletion mutants induced higher expression of the defence-related genes in banana plants. These results suggest that FocRnt2 plays an important role in full virulence of Foc TR4, further improving our understanding of effector-mediated Foc TR4 pathogenesis.

Streptomyces-Secreted Fluvirucin B6 as a Potential Bio-Fungicide for Managing Banana Fusarium Wilt and Mycotoxins and Modulating the Soil Microbial Community Structure.

Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc TR4) is the most destructive soil-borne fungal disease. Until now, there has been a lack of effective measures to control the disease. It is urgent to explore biocontrol agents to control Foc TR4 and the secretion of mycotoxin. In this study, fluvirucin B6 was screened from Streptomyces solisilvae using an activity-guided method. Fluvirucin B6 exhibited strong antifungal activity against Foc TR4 (0.084 mM of EC50 value) and significantly inhibited mycelial growth and spore germination. Further studies demonstrated that fluvirucin B6 could cause the functional loss of mitochondria, the disorder of metabolism of Foc TR4 cells, and the decrease of enzyme activities in the tricarboxylic acid cycle and electron transport chain, ultimately inhibiting mycotoxin metabolism. In a pot experiment, the application of fluvirucin B6 significantly decreased the incidence of banana Fusarium wilt and the amount of Foc TR4 and controlled fungal toxins in the soil. Additionally, fluvirucin B6 could positively regulate the changes in the structure of the banana rhizosphere microbial community, significantly enriching beneficial microbes associated with disease resistance. In summary, this study identifies fluvirucin B6, which plays versatile roles in managing fungal diseases and mycotoxins.

MaHOS15 negatively regulates plant immune defense induced by banana fusarium wilt via salicylic acid pathway

Fusarium wilt of banana (Musa acuminata) poses a significant threat to the banana industry globally. Salicylic acid (SA) serves as a crucial signaling molecule activating the plant's defense response against pathogen infections while also regulating various aspects of plant growth and development. High Expression of Osmotically Responsive Genes 15 (HOS15) acts as a transcriptional corepressor and an E3 ubiquitin ligase complex substrate receptor, playing an important role in regulating biological stress processes. However, the exact immune mechanism of MaHOS15 in the SA signaling pathway remains unclear. In this study, we utilized RT-qPCR, yeast two-hybridization, subcellular localization and allogeneic expression of Arabidopsis to explore MaHOS15's function against banana fusarium wilt. The expression changes of SA pathway-related factors MaNPR3, MaNPR4, MaNPR11, MaTGA8 and MaHOS15 in Foc TR4-resistant and Foc TR4-susceptible bananas infected with Foc TR4 were compared and analyzed. The results showed that the MaHOS15 expression trend in the two cultivars was opposite, while the expression trend of MaNPR4 and MaNPR11 was contrary to that of MaHOS15. Moreover, heterologous overexpression of MaHOS15 in Arabidopsis reduced the variance in SA-related signaling factors. The expression of MaHOS15 increased after defense activation, inhibiting the expression of SA-related immune factors, the immunoregulation of MaHOS15 and SA exhibited contrasting results. Collectively, these findings suggest that MaHOS15 negatively regulates plant immune defenses against banana Fusarium wilt via the salicylic acid pathway, offering potential applications in improving plant resistance to banana Fusarium wilt.

Determination of fermentation conditions of Streptomyces sp. VNUA116 to improve the antifungal activities against Fusarium oxysporum f.sp. cubense causing Panama wilt disease on banana plants

This study aims to identify medium components (carbon source, carbon concentration, nitrogen source) and fermentation conditions (temperature, pH) to enhance the resistance against Fusarium oxysporum f. sp. cubenseTropical Race 4 (Foc Tr4), causing Panama wilt disease of Streptomyces sp. VNUA116 (VNUA116). By using the agar well diffusion method, it was determined that strain VNUA116 has strong anti-fungal pathogen activities FocTr4 under fermentation nutrients: YIM 301 medium, carbon source CB1 (soluble starch 40 g/l: D-glucose 1 g/l, and nitrogen source N1 beef extract 3 g/l: yeast extract 5 g/l: peptone 3 g/l). Additionally, optimal fermentation conditions were established, including pH 7, and temperature 30oC. Strain VNUA116 exhibited the highest antifungal activity, reaching a diameter of 30.67±0.58 mm after 10 days of cultivation. Furthermore, under these optimised fermentation conditions, Streptomycessp. VNUA116 demonstrated an impressive inhibitory capability against Foc-Tr4, reaching up to 93.20%, equivalent to the concentration of the fungicide Ridomil gold 68WG at 3 g/l in laboratory conditions. This affirms the considerable potential of the VNUA116 strain for applications in the production of biofertilizers for the biological control of Panama wilt disease in banana plants.

A smooth vetch (Vicia villosa var.) strain endogenous to the broad-spectrum antagonist Bacillus siamensis JSZ06 alleviates banana wilt disease.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), poses a significant threat to banana production globally, thereby necessitating effective biocontrol methods to manage this devastating disease. This study investigates the potential of Bacillus siamensis strain JSZ06, isolated from smooth vetch, as a biocontrol agent against Foc TR4. To this end, we conducted a series of in vitro and in vivo experiments to evaluate the antifungal activity of strain JSZ06 and its crude extracts. Additionally, genomic analyses were performed to identify antibiotic synthesis genes, while metabolomic profiling was conducted to characterize bioactive compounds. The results demonstrated that strain JSZ06 exhibited strong inhibitory activity against Foc TR4, significantly reducing mycelial growth and spore germination. Moreover, scanning and transmission electron microscopy revealed substantial ultrastructural damage to Foc TR4 mycelia treated with JSZ06 extracts. Genomic analysis identified several antibiotic synthesis genes, and metabolomic profiling revealed numerous antifungal metabolites. Furthermore, in pot trials, the application of JSZ06 fermentation broth significantly enhanced banana plant growth and reduced disease severity, achieving biocontrol efficiencies of 76.71% and 79.25% for leaves and pseudostems, respectively. In conclusion, Bacillus siamensis JSZ06 is a promising biocontrol agent against Fusarium wilt in bananas, with its dual action of direct antifungal activity and plant growth promotion underscoring its potential for integrated disease management strategies.

Taxonomic identification and antagonistic activity of Streptomyces luomodiensis sp. nov. against phytopathogenic fungi.

Banana wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is a devastating fungal disease. Biocontrol strategies hold immense potential for inhibiting the spread of Foc TR4. Here, 30 actinobacteria were isolated from soils and screened for their antagonistic activity against Foc TR4. Strain SCA4-21T was selected due to its strongest antagonistic activity against Foc TR4. Strain SCA4-21T also exhibited strong antagonistic activity against the other eight phytopathogenic fungi. The strain was identified as the genus Streptomyces according to its physiological, biochemical, and phenotypic characteristics. The phylogenetic trees of 16S rRNA sequences demonstrated that strain SCA4-21T formed a subclade with S. iranensis HM 35T and/or S. rapamycinicus NRRL B-5491T with low bootstrap values. Considering that 16S rRNAs did not provide sufficient resolution for species-level identification, the whole genome of strain SCA4-21T was sequenced. Multilocus sequence analysis (MLSA) based on five housekeeping gene alleles (atpD, gyrB, recA, rpoB, and trpB) revealed that strain SCA4-21T clustered into S. hygroscopicus subsp. hygroscopicus NBRC 13472T with 100% of bootstrap value. The analysis of the genome-based phylogeny also approved the results. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were 91.26 and 44.30%, respectively, with values below the respective species level threshold of 95 and 70%. Hence, strain SCA 4-21T represented a novel species within the genus Streptomyces, named Streptomyces luomodiensis sp. nov. The type strain is SCA4-21T (=GDMCC4.340T = JCM36555T). By the CAZymes analysis, 348 carbohydrate-active enzymes (CAZymes) were detected, including 15 chitinases and eight β-1,3-glucanases. The fermentation broth of strain SCA4-21T, exhibiting strong antagonistic activity against Foc TR4, demonstrated high activities of chitinase and β-1,3-glucanase, which might be involved in antifungal activity. Our results showed an innovative potential biocontrol agent for managing plant fungal diseases, specifically banana fusarium wilt.

Molecular identification of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt disease of Tieu hong banana cultivar in the Red River Delta of Vietnam

The Red River Delta is one of primary banana-producing regions in Vietnam, and it has been afflicted by Fusarium wilt disease, caused by Fusarium oxysporum f. sp. cubense (Foc). This study aimed to identify Foc strains from banana-diseased samples collected from six different provinces in this region. Foc’s identity was confirmed based on morphological characteristics, artificial inoculation on the Tieu hong banana cultivar, and through DNA sequencing and phylogenetic analysis of PCR products amplified by race-specific primer pairs (Foc-1/Foc-2, Foc TR4-F/FocTR4-R). Ten Foc TR4 strains were identified, including VP1(2), VP1(5), and VP2(6) strains were found in Vinh Phuc Province, ML02.2 strain in Ha Noi City, MS1T2 strain in Hung Yen province, BV03 and BV06 strains in Hai Phong City, and QG1(R), QG4(T), QH2(R) strains in Thai Binh province. This discovery underscores the importance of appropriately identifying causal pathogens for integrated pest management of Fusarium wilt disease in the Red River Delta.

MaSMG7-Mediated Degradation of MaERF12 Facilitates Fusarium oxysporum f. sp. cubense Tropical Race 4 Infection in Musa acuminata.

Modern plant breeding relies heavily on the deployment of susceptibility and resistance genes to defend crops against diseases. The expression of these genes is usually regulated by transcription factors including members of the AP2/ERF family. While these factors are a vital component of the plant immune response, little is known of their specific roles in defense against Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) in banana plants. In this study, we discovered that MaERF12, a pathogen-induced ERF in bananas, acts as a resistance gene against Foc TR4. The yeast two-hybrid assays and protein-protein docking analyses verified the interaction between this gene and MaSMG7, which plays a role in nonsense-mediated RNA decay. The transient expression of MaERF12 in Nicotiana benthamiana was found to induce strong cell death, which could be inhibited by MaSMG7 during co-expression. Furthermore, the immunoblot analyses have revealed the potential degradation of MaERF12 by MaSMG7 through the 26S proteasome pathway. These findings demonstrate that MaSMG7 acts as a susceptibility factor and interferes with MaERF12 to facilitate Foc TR4 infection in banana plants. Our study provides novel insights into the biological functions of the MaERF12 as a resistance gene and MaSMG7 as a susceptibility gene in banana plants. Furthermore, the first discovery of interactions between MaERF12 and MaSMG7 could facilitate future research on disease resistance or susceptibility genes for the genetic improvement of bananas.

Characterisation of Streptomyces sp. VNUA116 with strong antifungal activity against Fusarium oxysporum f.sp. cubense tropical race 4

Banana (Musa spp.) is one of the most important crops in the world. However, banana production worldwide is threatened by the wilt disease caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Controlling this disease remains a challenge with conventional control methods, and scientists are searching for long-term, effective, and sustainable strategies. Streptomyces species are known as resources for producing bioactive compounds that represent promising bioagents to control soil-borne diseases. Here, we reported the characteristics of a Streptomyces strain, VNUA116, with strong antifungal activity against Foc TR4. The culture filtrate of the strain VNUA116 exhibited antifungal activities against Foc TR4 by inhibiting mycelial growth and spore germination. Interestingly, the strain VNUA116 also exhibited broad-spectrum antifungal activity against various common pathogenic fungi. Based on the morphological, biochemical, and molecular properties, the strain VNUA116 was identified as Streptomyces sp. In addition, Streptomyces sp.VNUA116 showed valuable productions of IAA, siderophore, and several hydrolytic enzyme activities. Moreover, the presence of the PKS-I and PKS-II genes in the genome of Streptomyces sp. VNUA116 suggests potential for the biosynthesis of bioactive substances. Our finding suggested that Streptomyces sp. VNUA116 could be a potential bioagent for controlling Fusarium wilt and other fungal diseases in bananas.

Unveiling the dynamic expression of PR-1 during Musa spp. infection by Fusarium oxysporum fsp. Cubense: a cloning and characterization study.

Pathogen-related proteins (PR) are pivotal in plant defense, combating diverse biotic and abiotic stresses. While multiple gene families contribute to banana resistance against Fusarium oxysporum f sp. cubense (Foc), Pseudocercospora eumusae, and Pratylenchus coffeae, the significance of PR-1 genes in defense is paramount. Three PR-1 genes, up-regulated under diverse biotic stresses, were cloned from both resistant and susceptible cultivars of Foc, P. eumusae, and P. coffeae. Molecular characterization, phylogenetic analysis, and docking studies with the Foc TR4 CP gene were conducted. Through transcriptomic and real-time studies, three PR-1 genes (Ma02_g15050, Ma02_g15060, and Ma04_g34800) from Musa spp. were identified. These genes exhibited significant up-regulation in resistant cultivars when exposed to Foc, P. eumusae, and P. coffeae. Cloning of these genes was successfully performed from both resistant and susceptible cultivars of Foc race 1 and TR4, P. eumusae, and P. coffeae. Distinct characteristics were observed among the PR-1 genes, with groups 1 and 2 being acidic with signal peptides, and group 3 being basic without signal peptides. All cloned PR-1 proteins belonged to the CAP superfamily (PF00188). Phylogenetic analysis revealed clustering patterns for acidic PR-1 proteins, and KEGG orthology showed associations with vital pathways, including MAPK signaling, plant hormone signal transduction, and plant-pathogen interaction. Secondary and tertiary structure analyses confirmed sequence conservation across studied species. Docking studies explored interactions between the cerato-platanin (CP) gene from Foc TR4 and Ma02_g15060 from banana, suggesting the potential hindrance of PR-1 antifungal activity through direct interaction. The findings underscore the crucial role of cloned PR-1 genes in banana plant defense mechanisms against a broad spectrum of biotic stresses. These genes, especially those in groups 1 and 2, hold promise as candidates for developing stress-tolerant banana cultivars. The study provides valuable insights into the molecular aspects of banana defense strategies, emphasizing the potential applications of PR-1 genes in enhancing banana resilience.

The Autophagy-Related Musa acuminata Protein MaATG8F Interacts with MaATG4B, Regulating Banana Disease Resistance to Fusarium oxysporum f. sp. cubense Tropical Race 4.

Banana is one of the most important fruits in the world due to its status as a major food source for more than 400 million people. Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes substantial losses of banana crops every year, and molecular host resistance mechanisms are currently unknown. We here performed a genomewide analysis of the autophagy-related protein 8 (ATG8) family in a wild banana species. The banana genome was found to contain 10 MaATG8 genes. Four MaATG8s formed a gene cluster in the distal part of chromosome 4. Phylogenetic analysis of ATG8 families in banana, Arabidopsis thaliana, citrus, rice, and ginger revealed five major phylogenetic clades shared by all of these plant species, demonstrating evolutionary conservation of the MaATG8 families. The transcriptomic analysis of plants infected with Foc TR4 showed that nine of the MaATG8 genes were more highly induced in resistant cultivars than in susceptible cultivars. Finally, MaATG8F was found to interact with MaATG4B in vitro (with yeast two-hybrid assays), and MaATG8F and MaATG4B all positively regulated banana resistance to Foc TR4. Our study provides novel insights into the structure, distribution, evolution, and expression of the MaATG8 family in bananas. Furthermore, the discovery of interactions between MaATG8F and MaATG4B could facilitate future research of disease resistance genes for the genetic improvement of bananas.

Biological Management of Panama Wilt of Banana Caused by Fusarium oxysporum f. sp. Cubense

Bananas are one of the most important fruit crops grown in tropical and sub-tropical regions of the world. It contributes to the livelihood of many growers and constitutes a larger proportion of fruit orchards in India. Panama wilt of banana is a major fungal disease affecting banana cultivations and farmers facing huge economic losses after the introduction of Fusarium oxysporum f.sp.cubense Foc TR4 strain B2 in Bihar .In the present investigation, native and commercial isolates of Trichoderma viride and Trichoderma harzianum are tested against the all isolates of Fusarium oxxysporum f.sp. cubense. Both the biocontrol agents were found highly effective against the Iso. M, Iso A and Iso.K under in vitro as well as under natural pot conditions. But in the case of Iso.G (Foc TR 4 strain B2), these bioagents were found moderately effective under in vivo and in vitro conditions.

Evaluation (Glasshouse conditions) of native fungal bioagents with multiple functions against Fusarium wilt disease (Foc TR4) in India

Evaluation (Glasshouse conditions) of native fungal bioagents with multiple functions against Fusarium wilt disease (Foc TR4) in India

Fungal Endophytes of Moringa (Moringa oleifera L.), Neem (Azadirachta indica) and Lavender (Lavandula angustifolia) and Their Biological Control of Fusarium Wilt of Banana

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), significantly affects the productivity of the banana crop in the field. Currently, there are no effective control measures available, and therefore, there is an urgent need to develop novel strategies to control the spread of the disease. Biological control is a promising strategy for the management and control of the disease. The aim of this study was to identify fungal endophytes associated with Moringa (Moringa oleifera), Neem tree (Azadirachta indica) and Lavender (Lavandula angustifolia) and their antifungal activities against Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). We isolated 69 fungal endophytes from different tissues of M. oleifera, A. indica and L. angustifolia and screened for antifungal activity against Foc TR4. Six fungal endophytes exhibited highest antagonistic activity against Foc TR4 based on dual-culture assays. Based on morphology and ITS gene sequence analysis, the selected six endophytes were identified to be related to Alternaria alternata (MB7 and NR3), Neofusicoccum parvum (LB1), Fusarium oxysporum (LR1), Talaromyces amestolkiae (MB14) and Alternaria tenuissima (NB6). The culture filtrates of the six fungal endophytes (LB1, LR1, MB7, MB14, NB6 and NR3) exhibited more than 50% inhibition of mycelial growth of Foc TR4 in vitro and were producers of β-1,3-glucanase. The six fungal endophytes showed biocontrol efficacy against Fusarium wilt in pot experiments. The findings from this study demonstrate that fungal endophytes LB1, LR1, MB7, MB14, NB6 and NR3 should be explored as biocontrol agents and biofertilizers in banana production.

Secondary metabolite induced tolerance to Fusarium oxysporum f.sp. cubense TR4 in banana cv. Grand Naine through in vitro bio-immunization: a prospective research translation from induction to field tolerance

An innovative tissue culture mediated incorporation of metabolite-based biomolecule (Bio-immune) at in vitro stage itself in banana cv. Grand Naine was developed and validated for the production of Fusarium oxysporum f.sp. cubense TR4 tolerant plantlets. The novel bio-immune formulation developed by us, exhibited a significant antifungal potency against Foc TR4 with a high percent inhibition (100%) at a 2.5% concentration of bio-immune on the 5th, 7th, and 9th DAI. Bio-immune integrated during in vitro shoot proliferation stage in banana cv. Grand Naine recorded significant enhancement in the growth of roots and shoots. Bio-immune (0.5%) fortified media produced 12.67 shoots per clump whereas control registered only 9.67 shoots per clump. Similarly, maximum root numbers (7.67) were observed in bio-immune plants which were significantly higher over control (5.0). The bio-immunized banana transplants recorded a higher survival rate (97.57%) during acclimatization as compared to the control (94.53%). Furthermore, evaluation of the bio-immunized plants in pot experiments revealed that unimmunized plants treated with FocTR4 (TF) exhibited mortality between 60 and 90 days. On the 90th day after planting, a high mean disease severity index (DSI) of 3.45 was observed with unimmunized plantlets while the bio-immunized plants (TFBI) and ICAR-FUSICONT treated plants (TFTR) showed substantially reduced DSI (0.20 and 1.00) compared to FocTR4 treated control (TF). Significant increases in polyphenol oxidase (PPO), peroxidase (POD), β-1,3-glucanase, phenylalanine ammonia-lyase (PAL), chitinase activities, and enhanced phenol contents were recorded in bio-immunized plants compared to unimmunized plants. Field experiments at two different locations in Bihar, India revealed that bunch weight, no. of hands/bunch, and no. of fingers/hand of bio-immune treated plants were significantly higher compared to the control.

Two haplotype-resolved genome assemblies for AAB allotriploid bananas provide insights into banana subgenome asymmetric evolution and Fusarium wilt control

Bananas (Musa spp.) are one of the world’s most important fruit crops and play a vital role in food security for many developing countries. Most banana cultivars are triploids derived from inter- and intraspecific hybridizations between the wild diploid ancestor species Musa acuminate (AA) and M. balbisiana (BB). We report two haplotype-resolved genome assemblies of the representative AAB-cultivated types, Plantain and Silk, and precisely characterize ancestral contributions by examining ancestry mosaics across the genome. Widespread asymmetric evolution is observed in their subgenomes, which can be linked to frequent homologous exchange events. We reveal the genetic makeup of triploid banana cultivars and verify that subgenome B is a rich source of disease resistance genes. Only 58.5% and 59.4% of Plantain and Silk genes, respectively, are present in all three haplotypes, with >50% of genes being differentially expressed alleles in different subgenomes. We observed that the number of upregulated genes in Plantain is significantly higher than that in Silk at one-week post-inoculation with Fusarium wilt tropical race 4 (Foc TR4), which confirms that Plantain can initiate defense responses faster than Silk. Additionally, we compared genomic and transcriptomic differences among the genes related to carotenoid synthesis and starch metabolism between Plantain and Silk. Our study provides resources for better understanding the genomic architecture of cultivated bananas and has important implications for Musa genetics and breeding.

Development and application of a droplet digital PCR assay for the detection of Fusarium oxysporum f. sp. cubense tropical race 4 from different biological samples

AbstractFusarium wilt of banana, caused by the pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a threat to food security as it affects the global banana industry. Control of this pathogen is complex due to the lack of resistant banana or plantain genotypes, lack of effective commercial fungicides, the production of chlamydospores, late appearance of symptoms in infected plants, high adaptability of the pathogen to diverse soil conditions and the polycyclic nature of the disease. Currently, the management strategy for Foc TR4 is exclusion and eradication, which is costly for the producer and the environment because the plants are treated with glyphosate and the affected area is quarantined for a long time. Thus, the development of early detection strategies for Foc TR4 from environmental samples and asymptomatic tissues with low inoculum concentration is essential; this would enable restriction of pathogen spread and minimize the environmental impact by eradication only in areas with accurate data of the pathogen's presence. In this study, three different PCR technologies (conventional PCR, quantitative PCR and droplet digital [dd] PCR) were evaluated for Foc TR4 detection in complex environmental samples. We report the development of a sensitive and specific ddPCR primer/probe set and protocols that can be used as a tool for Foc TR4 detection from symptomatic and asymptomatic plant tissue without prior DNA extraction. The limits of detection in drainage waters, footbaths and soil samples were evaluated using artificial inoculation assays. Finally, environmental samples from Foc TR4‐affected fields were analysed.

A newly isolated Trichoderma Parareesei N4-3 exhibiting a biocontrol potential for banana fusarium wilt by Hyperparasitism.

Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race4 (Foc TR4) is one of the most destructive soil-borne fungal diseases and currently threatens banana production around the world. Until now, there is lack of an effective method to control banana Fusarium wilt. Therefore, it is urgent to find an effective and eco-friendly strategy against the fungal disease. In this study, a strain of Trichoderma sp. N4-3 was isolated newly from the rhizosphere soil of banana plants. The isolate was identified as Trichoderma parareesei through analysis of TEF1 and RPB2 genes as well as morphological characterization. In vitro antagonistic assay demonstrated that strain N4-3 had a broad-spectrum antifungal activity against ten selected phytopathogenic fungi. Especially, it demonstrated a strong antifungal activity against Foc TR4. The results of the dual culture assay indicated that strain N4-3 could grow rapidly during the pre-growth period, occupy the growth space, and secrete a series of cell wall-degrading enzymes upon interaction with Foc TR4. These enzymes contributed to the mycelial and spore destruction of the pathogenic fungus by hyperparasitism. Additionally, the sequenced genome proved that strain N4-3 contained 21 genes encoding chitinase and 26 genes encoding β-1,3-glucanase. The electron microscopy results showed that theses cell wall-degrading enzymes disrupted the mycelial, spore, and cell ultrastructure of Foc TR4. A pot experiment revealed that addition of strain N4-3 significantly reduced the amount of Foc TR4 in the rhizosphere soil of bananas at 60 days post inoculation. The disease index was decreased by 45.00% and the fresh weight was increased by 63.74% in comparison to the control. Hence, Trichoderma parareesei N4-3 will be a promising biological control agents for the management of plant fungal diseases.