Tropical Race Research Articles

Fusarium wilt in banana: unraveling molecular aspects of host–pathogen interaction and resistance mechanism

Fusarium wilt in banana: unraveling molecular aspects of host–pathogen interaction and resistance mechanism

Deteksi Penyakit Layu Fusarium pada Pisang-Pisang Lokal di Pandeglang

Detection of Fusarium wilt on Local Bananas in Pandeglang
 Fusarium wilt of bananas or Panama disease, caused by Tropical Race4 (TR4), threatens the global banana industry. In Indonesia, the disease devastated Cavendish, an export variety, and many local bananas, such as Pisang Raja, Kepok, and Barangan. Nonetheless, TR4 incidence is unknown in many parts of Indonesia. This study aimed to detect TR4 in local bananas from Pandeglang, the most banana-produced area in Banten Province. A survey was conducted in nine Kecamatan i.e., Banjar, Cadasari, Jiput, Labuan, and Menes. In total, 13 banana accessions showed symptoms of fusarium wilt. Symptomatic pseudostem samples were plated on potato dextrose agar. The monosporic culture was derived from isolation and used for morphological identification and DNA extraction. Based on the morphological characteristics of Fusarium, 27 strains members of 3 species complexes were identified, i.e., F. cugenangense, F. foetens dan F. oxysporum (Fusarium oxysporum species complex; FOSC), F. equiseti dan F. incarnatum (Fusarium incarnatum-equiseti species complex) dan F. solani (Fusarium solani species complex, currently named as Neocosmospora). Molecular detection of TR4 was done using duplex PCR of translation elongation factor-1α (Tef-1α) and TR4 primer specific, giving positive results only on TR4 strains, members of FOSC. This study enhances our knowledge of the spreading of TR4 in an unidentified area in Indonesia. Many of the local bananas in Pandeglang is susceptible to TR4, thus threaten banana cultivation in this area. Further study on the molecular analysis using more genes will give insight into the expanding genetic diversity of Fusarium causing wilt in Banana in the Indonesian collection.

Screenhouse Evaluation of the Fungicides and Bio-Control Agents for the Management of Fusarium Wilt (Foc Race 1) of Banana in Nepal

Banana is affected by a wide number of diseases, of which, Fusarium wilt caused by Fusarium oxysporum f.sp. cubense has played a major role in devastating Malbhog (Silk) banana plantations in Nepal. The objective of this study was to evaluate the commonly available fungicides (Carbendazim 50%, Fenamidone 10%+ Mancozeb 50%, Chlorothalonil and biocontrol agents (Trichoderma sps., Bacillus thuringiensis and Pseudomonas species) for their efficacy against Foc. The effectiveness of fungicides and biocontrol agents to Foc was examined in screenhouse. The experiment was laid out in a completely randomized design with three replications and eight treatments. Among the chemicals, the most effective fungicide to reduce Fusarium wilt severity (root disease) was Bavistin with an efficacy of 55.33% followed by Sectin 46.33% and Chlorothalonil 35%. Similarly, maximum efficacy over control was recorded in Trichoderma harzianum with 41% and lowest was 32% in Pseudomonas sp. In the case of leaf disease, the lowest severity was 21.33% recorded in Bavistin and highest was 40.33% in Chlorothalonil. Similarly, among biocontrol agents, the lowest severity was found in Bacillus thuringiensis whereas highest was found in Trichoderma viride. A Suppression of disease by biocontrol agents might be due to their fungicidal activity, which means they produce a variety of antifungal compounds. These compounds induce plant defense mechanisms. Further field evaluations of the fungicides and biocontrol agents are required to determine if the effect observed in the screenhouse can be integrated into field management of Fusarium wilt.

Mapping of the Susceptibility of Colombian Musaceae Lands to a Deadly Disease: Fusarium oxysporum f. sp. cubense Tropical Race 4

Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) (Syn. Fusarium odoratissimum) is a devastating soil-borne pathogen that infects the roots of banana plants and causes Fusarium wilt disease. Colombia is one of the world’s leading banana producers; therefore, new uncontrolled outbreaks could have serious consequences. Despite this, little is known about the susceptibility of Musaceae lands in Colombia to Foc TR4. This work presents a pioneering study on the susceptibility of Colombian soils to Foc TR4. For this, a study was carried out to characterize climatic, edaphic, and density factors of Musaceae productive systems at the Colombian level, articulated with expert criteria to map and define areas with different levels of susceptibility to Foc R4T. These criteria are typically selected based on the existing scientific literature, consultation with domain experts, and consideration of established methods for assessing soil health and disease susceptibility in Musaceae plantations. By joining the analyzed susceptibility factors, differentiated areas were generated that imply a greater or lesser predisposition to the disease. Subsequently, a validation of the classification was made with Random Forest. The results indicate that at the level of climate, soil, and farm density as a fit factor, practically 50% of the cultivated territory of Musaceae are areas high and very highly susceptible to the pathogen (572,000 km2). The results showed that from the total Musaceae area, Antioquia, Bolívar, Chocó, and Santander turned out to be the departments with the highest proportion of very high susceptibility class of the production farms. The analysis of Random Forest classification performance shows that the model has a relatively low out-of-bag (OOB) error rate (0.023). The study on the susceptibility is highly novel and original, as it represents the first systematic investigation of Foc TR4 susceptibility in Colombian soils. This paper provides important insights into the susceptibility of Musaceae lands in Colombia to Foc TR4. The study highlights the need for ongoing monitoring, containment, and control measures to prevent the spread of this deadly pathogen and protect Colombia’s important banana industry.

Iron necessity for chlamydospore germination in Fusarium oxysporum f. sp. cubense TR4.

Fusarium wilt disease of banana, caused by the notorious soil-borne pathogen Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), is extremely difficult to manage. Manipulation of soil pH or application of synthetic iron chelators can suppress the disease through iron starvation, which inhibits the germination of pathogen propagules called chlamydospores. However, the effect of iron starvation on chlamydospore germination is largely unknown. In this study, scanning electron microscopy was used to assemble the developmental sequence of chlamydospore germination and to assess the effect of iron starvation and pH in vitro. Germination occurs in three distinct phenotypic transitions (swelling, polarized growth, outgrowth). Outgrowth, characterized by formation of a single protrusion (germ tube), occurred at 2 to 3h, and a maximum value of 69.3% to 76.7% outgrowth was observed at 8 to 10h after germination induction. Germination exhibited plasticity with pH as over 60% of the chlamydospores formed a germ tube between pH 3 and pH 11. Iron-starved chlamydospores exhibited polarized-growth arrest, characterized by the inability to form a germ tube. Gene expression analysis of rnr1 and rnr2, which encode the iron-dependent enzyme ribonucleotide reductase, showed that rnr2 was upregulated (p < 0.0001) in iron-starved chlamydospores compared to the control. Collectively, these findings suggest that iron andextracellular pH arecrucial for chlamydospore germination in Foc TR4. Moreover, inhibition of germination by iron starvation may be linked to a different mechanism, rather than repression of the function of ribonucleotide reductase, the enzyme that controls growth by regulation of DNA synthesis.

Validation and Application of a Molecular Detection System for Fusarium Wilt of Banana in China.

Fusarium wilt of banana is a devastating disease caused by Fusarium oxysporum f. sp. cubense (Foc). It has restricted the development of the banana industry worldwide and is particularly serious in China because of the large planting areas and special planting patterns. However, there is no rapid and accurate approach to detect the Foc strains that specifically occur in China because of the rich genetic diversity observed in this pathosystem. In this study, we evaluated the performance of 10 previously published PCR primer pairs on 103 representative Foc strains in China and neighboring countries and screened out a set of primers (Foc-specific primer pair SIX9-Foc-F/R, Foc R1-specific primer pair SIX6b-210-F/R, Foc R4-specific primer pair Foc-1/2, and Foc TR4-specific primer pair W2987F/R) suitable for the detection of Foc strains in China and the surrounding Southeast Asian countries. Moreover, we developed a molecular detection system to accurately identify the different physiological races of Foc. The findings of this study provide technical support for preventing and controlling the spread of Fusarium wilt of banana in the field in China.

The Vulnerability of Cuban Banana Production to Fusarium Wilt Caused by Tropical Race 4.

Bananas are major agricultural commodities in Cuba. One of the main constraints of banana production worldwide is Fusarium wilt of banana. Recent outbreaks in Colombia, Perú, and Venezuela have raised widespread concern in Latin America due to the potential devastating impact on the sustainability of banana production, food security, and livelihoods of millions of people in the region. Here, we phenotyped 18 important Cuban banana and plantain varieties with two Fusarium strains-Tropical Race 4 (TR4) and Race 1-under greenhouse conditions. These varieties represent 72.8% of the national banana acreage in Cuba and are also widely distributed in Latin America and the Caribbean region. A broad range of disease responses from resistant to very susceptible was observed against Race 1. On the contrary, not a single banana variety was resistant to TR4. These results underscore that TR4 potentially threatens nearly 56% of the contemporary Cuban banana production area, which is planted with susceptible and very susceptible varieties, and call for a preemptive evaluation of new varieties obtained in the national breeding program and the strengthening of quarantine measures to prevent the introduction of TR4 into the country.

Antimicrobial mechanisms and secondary metabolite profiles of Streptomyces hygroscopicus subsp. hygroscopicus 5-4 against banana fusarium wilt disease using metabolomics.

Fusarium wilt of bananas (FWB) is seriously affecting the sustainable development of the banana industry and is caused by the devastating soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Biological control is a promising strategy for controlling Fusarium wilt in bananas. We previously identified Streptomyces hygroscopicus subsp. hygroscopicus 5-4 with strong antifungal activity against the FWB. The most possible antimicrobial mechanism of strain 5-4 was explored using the metabolomics approach, light microscopy imaging, and transmission electron microscopy (TEM). The membrane integrity and ultrastructure of Foc TR4 was damaged after extract treatment, which was supported by the degradation of mycelium, soluble protein content, extracellular reducing sugar content, NADH oxidase activity, malondialdehyde content, mitochondrial membrane potential, and mitochondrial respiratory chain complex enzyme activity. The extracts of strain 5-4 cultivated at different times were characterized by a liquid chromatography-mass spectrometer (LC-MS). 647 known metabolites were detected in the extracts of strains 5-4. Hygromycin B, gluten exorphin B4, torvoside G, (z)-8-tetradecenal, piperitoside, sarmentosin, pubescenol, and other compounds were the main differential metabolites on fermentation culture for 7 days. Compared with strain 5-4 extracts, hygromycin B inhibited the mycelial growth of Foc TR4, and the EC50 concentration was 7.4 μg/mL. These results showed that strain 5-4 could destroy the cell membrane of Foc TR4 to inhibit the mycelial growth, and hygromycin B may be the key antimicrobial active metabolite. Streptomyces hygroscopicus subsp. hygroscopicus 5-4 might be a promising candidate strain to control the FWB and provide a scientific basis for the practical application of hygromycin B as a biological control agent.

Genetic Mapping, Candidate Gene Identification and Marker Validation for Host Plant Resistance to the Race 4 of Fusarium oxysporum f. sp. cubense Using Musa acuminata ssp. malaccensis.

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. Cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. Malaccensis segregating populations, segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of 'DH-Pahang' reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F2 progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent 'Ma850' and a susceptible line 'Ma848', to show that the STR4 resistance co-segregated with marker '28820' at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as 'Pahang', 'SH-3362', 'SH-3217', 'Ma-ITC0250', and 'DH-Pahang/CIRAD 930'. Additional screening in the International Institute for Tropical Agriculture's collection suggests that the dominant allele is common among the elite 'Matooke' NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.

Antifungal Potential of Melaleuca alternifolia against Fungal Pathogen Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium wilt of bananas caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) poses the most serious threat to banana production globally. The disease has been managed using chemical fungicides, yet the control levels are still unsatisfactory. This study investigated the antifungal activities of tea tree (Melaleuca alternifolia) essential oil (TTO) and hydrosol (TTH) against Foc TR4 and their bioactive components. The potential of TTO and TTH in inhibiting the growth of Foc TR4 was evaluated in vitro using agar well diffusion and spore germination assays. Compared to the chemical fungicide, TTO effectively suppressed the mycelial growth of Foc TR4 at 69%. Both the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of TTO and TTH were established at 0.2 µg/µL and 50% v/v, respectively, suggesting the fungicidal nature of the plant extracts. The disease control efficacies were also demonstrated by a (p ≤ 0.05) delayed Fusarium wilt symptom development in the susceptible banana plants with reduced LSI dan RDI scores from 70% to around 20-30%. A GC/MS analysis of TTO identified terpinen-4-ol, eucalyptol, and α-terpineol as the major components. In contrast, an LC/MS analysis of TTH identified different compounds, including dihydro-jasmonic acid and methyl ester. Our findings indicate the potential of tea tree extracts as natural alternatives to chemical fungicides to control Foc TR4.

Revealing the different resistance mechanisms of banana ‘Guijiao 9’ to Fusarium oxysporum f. sp. cubense tropical race 4 using comparative proteomic analysis

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense is a worldwide devastating fungal disease in the banana industry. The disease caused by Fusarium oxysporum f. sp. cubense is becoming more and more serious. The pathogen of Fusarium oxysporum f. sp. cubense tropical race 4 (Foc4) is the most harmful one. ‘Guijiao 9’ is a banana cultivar with good resistance to Foc4, which is identified by resistance screening of natural variant lines. It is of great significance to explore the resistance genes and key proteins of ‘Guijiao 9’ for banana cultivar improvement and disease resistance breeding. In this study, iTRAQ (isobaric Tags for Relative and Absolute quantitation) was used to analyze the xylem proteomic data of banana roots from the resistant variety ‘Guijiao 9’ and susceptible variety ‘Williams’, and the differences in protein accumulation profiles between these two varieties at 24, 48, and 72 h after infection with Foc4 were compared. The identified proteins were analyzed by the protein WGCNA (Weighted Gene Correlation Network Analysis), and the differentially expressed proteins (DEPs) were verified by qRT-PCR experiments. Proteomic analysis showed that there were differences in the protein accumulation profiles of the resistant cultivar ‘Guijiao 9’ and the susceptible cultivar ‘Williams’ after infection with Foc4, and there were differences in resistance-related proteins, biosynthesis of secondary metabolites, peroxidase, and pathogenesis-related proteins. The stress response of bananas to pathogens was affected by multiple factors. Protein co-expression analysis showed that there was a high correlation between the MEcyan module and resistance, and ‘Guijiao 9’ had a different resistance mechanism compared with ‘Williams’. Significance‘Guijiao 9’ is a banana variety with good resistance to Foc4, which is identified by screening the resistance of natural variant lines in the farmland where banana plants are seriously infected by Foc4. It is of great significance to excavate the resistance genes and key proteins of ‘Guijiao 9’ for banana variety improvement and disease resistance breeding. The aim of this paper is to identify the proteins and related functional modules controlling the pathogenicity differences of Foc4 by comparative proteomic analysis of ‘Guijiao 9’, so as to understand the resistance mechanism of banana to Fusarium wilt, and offer basis for the final identification, isolation and utilization of Foc4 resistance-related genes in banana variety improvement. The research results will also provide a basis for further understanding the host-pathogen interaction and revealing the resistance mechanism of bananas.

Isolation and characterization of extracellular vesicles from Fusarium oxysporum f. sp. cubense, a banana wilt pathogen

Fusarium wilt of banana is a destructive widespread disease caused by Fusarium oxysporum f. sp. cubense (Foc) that ravaged banana plantations globally, incurring huge economic losses. Current knowledge demonstrates the involvement of several transcription factors, effector proteins, and small RNAs in the Foc-banana interaction. However, the precise mode of communication at the interface remains elusive. Cutting-edge research has emphasized the significance of extracellular vesicles (EVs) in trafficking the virulent factors modulating the host physiology and defence system. EVs are ubiquitous inter- and intra-cellular communicators across kingdoms. This study focuses on the isolation and characterization of Foc EVs from methods that make use of sodium acetate, polyethylene glycol, ethyl acetate, and high-speed centrifugation. Isolated EVs were microscopically visualized using Nile red staining. Further, the EVs were characterized using transmission electron microscopy, which revealed the presence of spherical, double-membrane, vesicular structures ranging in size from 50 to 200 nm (diameter). The size was also determined using the principle based on Dynamic Light Scattering. The Foc EVs contained proteins that were separated using SDS-PAGE and ranged between 10 and 315 kDa. Mass spectrometry analysis revealed the presence of EV-specific marker proteins, toxic peptides, and effectors. The Foc EVs were found to be cytotoxic, whose toxicity increased with EVs isolated from the co-culture preparation. Taken together, a better understanding of Foc EVs and their cargo will aid in deciphering the molecular crosstalk between banana and Foc.

Metatranscriptome-based strategy reveals the existence of novel mycoviruses in the plant pathogenic fungus Fusarium oxysporum f. sp. cubense.

Fusarium oxysporum f. sp. cubense (Foc) is a devastating plant pathogen that caused a great financial loss in the banana's source area. Metatranscriptomic analysis was used to determine the diversity of mycoviruses in 246 isolates of F. oxysporum f. sp. cubense. Partial or nearly complete genomes of 20 mycoviruses were obtained by BLASTp analysis of RNA sequences using the NCBI database. These 20 viruses were grouped into five distinct lineages, namely Botourmiaviridae, Endornaviridae, Mitoviridae, Mymonaviridae, Partitiviridae, and two non-classified mycoviruses lineages. To date, there is no report of the presence of mycoviruses in this pathogen. In this study, we demonstrate the presence of mycoviruses isolated from Foc. These findings enhance our overall knowledge of viral diversity and taxonomy in Foc. Further characterization of these mycoviruses is warranted, especially in terms of exploring these novel mycoviruses for innovative biocontrol of banana Fusarium wilt disease.

Distribution and diversity of Fusarium oxysporum f.sp. cubense TR4 causing banana wilt in Sub-tropics of India and comparative analysis of TR4 specific molecular detection methods

Fusarium wilt caused by Fusarium oxysporum f.sp. cubense (Foc) is regarded as the most recurring and deadliest threat to banana cultivation worldwide. Among three physiological races of the pathogen capable of infecting Musa spp., Tropical race 4 (TR4) is considered the most virulent race of Foc without any known source for resistance. In India, race 1 was commonly observed to affect the cultivars of Pisang Awak, Rasthali (Silk), and Malbhog (Silk) while the Grand Naine (G9) the most popular and extensively grown cultivar of Cavendish banana in subtropics of Uttar Pradesh (U.P.) and Bihar was free from the disease till 2018. But in 2018, the incidence of wilt was first time observed in Sohawal, Ayodhya (U.P.) later in Katihar (Bihar) of India. The Association of TR4 was later confirmed with diseased samples. In the current study, the distribution and incidence of TR4 was assessed in the subtropics covering the states of U.P. and Bihar. Forty-one isolates pathogenic to the banana plant were collected from 6 states, which varied in terms of aggressiveness, and morphological characteristics like colony color, spore size, and shape. The isolates from U.P. and Bihar were characterized as TR4 using two primer sets targeting the SIX1a (secreted in the xylem) gene however isolates from Gujarat, Assam, Andhra Pradesh, and Tamil Nadu (TN) were identified as Race 1 using two primer sets targeting SIX6b and a hypothetical protein (XM_018394505.1). Genetic variability was assessed using 8 ISSR markers and divided all 41 isolates into two major groups, with a set of isolates from the U.P. and Bihar in group A and isolates from other states in group B. In the comparative analysis of three TR4-specific primers, only 1 out of 3 primer sets was confirmed to be more accurate in the characterization of the TR4. Analysis of molecular variance revealed that genetic variations among the regions and within the region accounted for 37.0 per cent and 63.0 per cent. The current study signified that the spread of the TR4 in U.P. and Bihar suggests the need for efforts for management with emphasis on biosecurity practices and the use of antagonistic biological agents to prevent further spread.

Natural beneficial microorganisms can suppress Fusarium wilt of banana and have the potential for wider use in organic production systems

Natural beneficial microorganisms can suppress Fusarium wilt of banana and have the potential for wider use in organic production systems

Antifungal effects and biocontrol potential of lipopeptide-producing Streptomyces against banana Fusarium wilt fungus Fusarium oxysporum f. sp. cubense.

Fusarium wilt of banana (FWB), caused by Fusarium oxysporum f. sp. cubense (Foc), especially tropical race 4 (TR4), presents the foremost menace to the global banana production. Extensive efforts have been made to search for efficient biological control agents for disease management. Our previous study showed that Streptomyces sp. XY006 exhibited a strong inhibitory activity against several phytopathogenic fungi, including F. oxysporum. Here, the corresponding antifungal metabolites were purified and determined to be two cyclic lipopeptide homologs, lipopeptin A and lipopeptin B. Combined treatment with lipopeptin complex antagonized Foc TR4 by inhibiting mycelial growth and conidial sporulation, suppressing the synthesis of ergosterol and fatty acids and lowering the production of fusaric acid. Electron microscopy observation showed that lipopeptide treatment induced a severe disruption of the plasma membrane, leading to cell leakage. Lipopeptin A displayed a more pronounced antifungal activity against Foc TR4 than lipopeptin B. In pot experiments, strain XY006 successfully colonized banana plantlets and suppressed the incidence of FWB, with a biocontrol efficacy of up to 87.7%. Additionally, XY006 fermentation culture application improved plant growth parameters and induced peroxidase activity in treated plantlets, suggesting a possible role in induced resistance. Our findings highlight the potential of strain XY006 as a biological agent for FWB, and further research is needed to enhance its efficacy and mode of action in planta.

Loss of Phosphomannose Isomerase Impairs Growth, Perturbs Cell Wall Integrity, and Reduces Virulence of Fusarium oxysporum f. sp. cubense on Banana Plants.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana, necessitating urgent measures to control this disease. However, the molecular mechanisms underlying Foc TR4 virulence remain elusive. Phosphomannose isomerase is a key enzyme involved in the biosynthesis of GDP mannose, an important precursor of fungal cell walls. In this study, two phosphomannose isomerases were identified in the Foc TR4 genome, of which only Focpmi1 was highly expressed throughout all developmental stages. Generated null mutants in Foc TR4 showed that only the ΔFocpmi1 mutant required exogenous mannose for growth, indicating that Focpmi1 is the key enzyme involved in GDP mannose biosynthesis. The Focpmi1 deficient strain was unable to grow without exogenous mannose and exhibited impaired growth under stress conditions. The mutant had reduced chitin content in its cell wall, rendering it vulnerable to cell wall stresses. Transcriptomic analysis revealed up- and down-regulation of several genes involved in host cell wall degradation and physiological processes due to the loss of Focpmi1. Furthermore, Focpmi1 was also found to be crucial for Foc TR4 infection and virulence, making it a potential antifungal target to address the threats posed by Foc TR4.

A Novel Effector, FSE1, Regulates the Pathogenicity of Fusarium oxysporum f. sp. cubense Tropical Race 4 to Banana by Targeting the MYB Transcription Factor MaEFM-Like.

Phytopathogenic fungi secretes a range of effectors to manipulate plant defenses. Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is a soil-borne pathogen that causes destructive banana wilt disease. Understanding the molecular mechanisms behind Foc TR4 effectors and their regulation of pathogenicity is helpful for developing disease control strategies. In the present study, we identified a novel effector, Fusarium special effector 1 (FSE1), in Foc TR4. We constructed FSE1 knock-out and overexpression mutants and investigated the functions of this effector. In vitro assays revealed that FSE1 was not required for vegetative growth and conidiation of Foc TR4. However, inoculation analysis of banana plantlets demonstrated that knock-out of FSE1 increased the disease index, while overexpression of FSE1 decreased it. Microscope analysis suggested that FSE1 was distributed in the cytoplasm and nuclei of plant cells. Furthermore, we identified an MYB transcription factor, MaEFM-like, as the target of FSE1, and the two proteins physically interacted in the nuclei of plant cells. In addition, Transient expression of MaEFM-like induced cell death in tobacco leaves. Our findings suggest that FSE1 is involved in the pathogenicity of Foc TR4 by targeting MaEFM-like.

Characterization of Banana SNARE Genes and Their Expression Analysis under Temperature Stress and Mutualistic and Pathogenic Fungal Colonization.

SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors) are engines for almost all of the membrane fusion and exocytosis events in organism cells. In this study, we identified 84 SNARE genes from banana (Musa acuminata). Gene expression analysis revealed that the expression of MaSNAREs varied a lot in different banana organs. By analyzing their expression patterns under low temperature (4 °C), high temperature (45 °C), mutualistic fungus (Serendipita indica, Si) and fungal pathogen (Fusarium oxysporum f. sp. Cubense Tropical Race 4, FocTR4) treatments, many MaSNAREs were found to be stress responsive. For example, MaBET1d was up-regulate by both low and high temperature stresses; MaNPSN11a was up-regulated by low temperature but down-regulated by high temperature; and FocTR4 treatment up-regulated the expression of MaSYP121 but down-regulated MaVAMP72a and MaSNAP33a. Notably, the upregulation or downregulation effects of FocTR4 on the expression of some MaSNAREs could be alleviated by priorly colonized Si, suggesting that they play roles in the Si-enhanced banana wilt resistance. Foc resistance assays were performed in tobacco leaves transiently overexpressing MaSYP121, MaVAMP72a and MaSNAP33a. Results showed that transient overexpression of MaSYP121 and MaSNPA33a suppressed the penetration and spread of both Foc1 (Foc Race 1) and FocTR4 in tobacco leaves, suggesting that they play positive roles in resisting Foc infection. However, the transient overexpression of MaVAMP72a facilitated Foc infection. Our study can provide a basis for understanding the roles of MaSNAREs in the banana responses to temperature stress and mutualistic and pathogenic fungal colonization.

Fusarium mindanaoense sp. nov., a New Fusarium Wilt Pathogen of Cavendish Banana from the Philippines Belonging to the F. fujikuroi Species Complex.

The pathogen causing Fusarium wilt in banana is reported to be Fusarium oxysporum f. sp. cubense (FOC). In 2019, wilt symptoms in banana plants (cultivar: Cavendish) in the Philippines were detected, i.e., the yellowing of the leaves and discoloration of the pseudostem and vascular tissue. The fungus isolated from the vascular tissue was found to be pathogenic to Cavendish bananas and was identified as a new species, F. mindanaoense, belonging to the F. fujikuroi species complex (FFSC); species classification was assessed using molecular phylogenetic analyses based on the tef1, tub2, cmdA, rpb1, and rpb2 genes and morphological analyses. A reciprocal blast search using genomic data revealed that this fungus exclusively included the Secreted in Xylem 6 (SIX6) gene among the SIX homologs related to pathogenicity; it exhibited a highly conserved amino acid sequence compared with that of species in the FFSC, but not with that of FOC. This was the first report of Fusarium wilt in Cavendish bananas caused by a species of the genus Fusarium other than those in the F. oxysporum species complex.