Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4.
ABSTRACT: Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics.
Project description:Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel subgroup succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial subgroup worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to cultivars of Cavendish, and other commonly grown cultivars. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana cultivars. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability, and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, and TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas.
Project description:Fusarium oxysporum formae specialis cubense (Foc) is a soil-borne fungus that causes Fusarium wilt, which is considered to be the most destructive disease of bananas. The fungus is believed to have evolved with its host in the Indo-Malayan region, and from there it was spread to other banana-growing areas with infected planting material. The diversity and distribution of Foc in Asia was investigated. A total of 594 F. oxysporum isolates collected in ten Asian countries were identified by vegetative compatibility groups (VCGs) analysis. To simplify the identification process, the isolates were first divided into DNA lineages using PCR-RFLP analysis. Six lineages and 14 VCGs, representing three Foc races, were identified in this study. The VCG complex 0124/5 was most common in the Indian subcontinent, Vietnam and Cambodia; whereas the VCG complex 01213/16 dominated in the rest of Asia. Sixty-nine F. oxysporum isolates in this study did not match any of the known VCG tester strains. In this study, Foc VCG diversity in Bangladesh, Cambodia and Sri Lanka was determined for the first time and VCGs 01221 and 01222 were first reported from Cambodia and Vietnam. New associations of Foc VCGs and banana cultivars were recorded in all the countries where the fungus was collected. Information obtained in this study could help Asian countries to develop and implement regulatory measures to prevent the incursion of Foc into areas where it does not yet occur. It could also facilitate the deployment of disease resistant banana varieties in infested areas.
Project description:Banana is the most popular and most exported fruit and also a major food crop for millions of people around the world. Despite its importance and the presence of serious disease threats, research into this crop is limited. One of those is Panama disease or Fusarium wilt. In the previous century Fusarium wilt wiped out the "Gros Michel" based banana industry in Central America. The epidemic was eventually quenched by planting "Cavendish" bananas. However, 50 years ago the disease recurred, but now on "Cavendish" bananas. Since then the disease has spread across South-East Asia, to the Middle-East and the Indian subcontinent and leaped into Africa. Here, we report the presence of Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4) in "Cavendish" plantations in Laos, Myanmar, and Vietnam. A combination of classical morphology, DNA sequencing, and phenotyping assays revealed a very close relationship between the Foc TR4 strains in the entire Greater Mekong Subregion (GMS), which is increasingly prone to intensive banana production. Analyses of single-nucleotide polymorphisms enabled us to initiate a phylogeography of Foc TR4 across three geographical areas-GMS, Indian subcontinent, and the Middle East revealing three distinct Foc TR4 sub-lineages. Collectively, our data place these new incursions in a broader agroecological context and underscore the need for awareness campaigns and the implementation of validated quarantine measures to prevent further international dissemination of Foc TR4.
Project description:Banana is an important food crop and source of income in Africa. Sustainable production of banana, however, is at risk because of pests and diseases such as Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). Foc can be disseminated from infested to disease-free fields in plant material, water and soil. Early detection of Foc using DNA technologies is thus required to accurately identify the fungus and prevent its further dissemination with plants, soil and water. In this study, quantitative (q)PCR assays were developed for the detection of Foc Lineage VI strains found in central and eastern Africa (Foc races 1 and 2), Foc TR4 (vegetative compatibility groups (VCG) 01213/16) that is present in Mozambique, and Foc STR4 (VCG 0120/15) that occurs in South Africa. A collection of 127 fungal isolates were selected for specificity testing, including endophytic Fusarium isolates from banana pseudostems, non-pathogenic F. oxysporum strains and Foc isolates representing the 24 VCGs in Foc. Primer sets that proved to be specific to Foc Lineage VI, Foc TR4 and Foc STR4 were used to produce standard curves for absolute quantification, and the qPCR assays were evaluated based on the quality of standard curves, repeatability and reproducibility, and limits of quantification (LOQ) and detection (LOD). The qPCR assays for Foc Lineage VI, TR4 and STR4 were repeatable and reproducible, with LOQ values of 10-3-10-4 ng/?L and a LOD of 10-4-10-5 ng/?L. The quantitative detection of Foc strains in Africa could reduce the time and improve the accuracy for identifying the Fusarium wilt pathogen from plants, water and soil on the continent.
Project description:Bananas are vital for food security in many countries, and half of banana production relies solely on 'Cavendish' (AAA), which is presently threatened by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) tropical race 4. This particular virulent Foc strain was also found to attack other banana varieties of commercial importance. As there is no single effective management practice available so far, this study was undertaken to determine resistant sources from the genotype collection available at the ICAR-National Research Centre for Banana, Tiruchirappalli, Tamil Nadu, India for direct use by farmers and/or in breeding programmes to develop resistant hybrids. A total of 258 genotypes of different ploidies and genomic constitutions were tested against Foc race 1 (VCG 0124). In total, 19 genotypes (AA Unique-6, BB type-2, AAA Unique-1, AAA Cavendish-1, AAB Mysore-3, AAB Pome-1, AAB Plantain-4 and AAAB-1) were found to be immune; eight genotypes (AA Unique-1, BB type-3, AAA Cavendish-1, AAB Mysore-1, AAB Unique-1, AAB Plantain-1) were highly resistant; and nine genotypes (AA Unique-1, AAA Cavendish-3, AAB Silk-1, AAB Pome-4) were resistant. The genotypes that are resistant to the virulent Foc race 1 (VCG 0124) strain can be exploited directly for commercialization and/or in breeding programs to develop resistant hybrids.
Project description:BACKGROUND: Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is considered the most lethal disease of Cavendish bananas in the world. The disease can be managed in the field by planting resistant Cavendish plants generated by somaclonal variation. However, little information is available on the genetic basis of plant resistance to Foc TR4. To a better understand the defense response of resistant banana plants to the Fusarium wilt pathogen, the transcriptome profiles in roots of resistant and susceptible Cavendish banana challenged with Foc TR4 were compared. RESULTS: RNA-seq analysis generated more than 103 million 90-bp clean pair end (PE) reads, which were assembled into 88,161 unigenes (mean size = 554 bp). Based on sequence similarity searches, 61,706 (69.99%) genes were identified, among which 21,273 and 50,410 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 33,243 (37.71%) unigenes to 119 KEGG pathways. A total of 5,008 genes were assigned to plant-pathogen interactions, including disease defense and signal transduction. Digital gene expression (DGE) analysis revealed large differences in the transcriptome profiles of the Foc TR4-resistant somaclonal variant and its susceptible wild-type. Expression patterns of genes involved in pathogen-associated molecular pattern (PAMP) recognition, activation of effector-triggered immunity (ETI), ion influx, and biosynthesis of hormones as well as pathogenesis-related (PR) genes, transcription factors, signaling/regulatory genes, cell wall modification genes and genes with other functions were analyzed and compared. The results indicated that basal defense mechanisms are involved in the recognition of PAMPs, and that high levels of defense-related transcripts may contribute to Foc TR4 resistance in banana. CONCLUSIONS: This study generated a substantial amount of banana transcript sequences and compared the defense responses against Foc TR4 between resistant and susceptible Cavendish bananas. The results contribute to the identification of candidate genes related to plant resistance in a non-model organism, banana, and help to improve the current understanding of host-pathogen interactions.
Project description:Fusarium oxysporum f.sp. cubense (Foc) is considered one of the most devastating soilborne fungal pathogens of banana worldwide. Foc causing mortality to Cavendish group bananas, and belonging to the unique vegetative compatibility group (VCG) 01213/16 has been termed tropical race 4 (TR4) and has currently been renamed F. odoratissimum. The pathogen that was first detected approximately 50 years ago in South East Asia, has since spread to countries within the greater Mekong subregion and to Australia. Recently, the pathogen disseminated to India, Pakistan, Oman and Mozambique (Africa) and was identified in the South American continent in Colombia in 2019. In the Middle East, TR4 was first reported from Jordan and Lebanon, and later from Israel in 2016. In Israel, the pathogen was identified as TR4 by VCG tests, pathogenicity assays and molecular verification. The complete genomes of five representative TR4 isolates including two from Israel, one from Jordan, one from the Philippines, and one from Indonesia were sequenced, and single nucleotide polymorphisms (SNPs) analyses were conducted. SNPs were compared to 11 additional sequenced TR4 isolates, to determine the origin of the Israeli isolates. SNP detection and phylogeographical analyses determined that the Middle Eastern isolates are closely related, indicating that the pathogen most likely spread to Israel from Jordan, while those from Colombia are related to a representative isolate from Indonesia.
Project description:The continued dispersal of Fusarium oxysporum f. sp. cubense Tropical race 4 (FocTR4), a quarantine soil-borne pathogen that kills banana, has placed this worldwide industry on alert and triggered enormous pressure on National Plant Protection (NPOs) agencies to limit new incursions. Accordingly, biosecurity plays an important role while long-term control strategies are developed. Aiming to strengthen the contingency response plan of Ecuador against FocTR4, a population biology study-including phylogenetics, mating type, vegetative compatibility group (VCG), and pathogenicity testing-was performed on isolates affecting local bananas, presumably associated with race 1 of F. oxysporum f. sp. cubense (Foc). Our results revealed that Foc populations in Ecuador comprise a single clonal lineage, associated with VCG0120. The lack of diversity observed in Foc populations is consistent with a single introduction event from which secondary outbreaks originated. The predominance of VCG0120, together with previous reports of its presence in Latin America countries, suggests this group as the main cause of the devastating Fusarium wilt epidemics that occurred in the 1950s associated to the demise of 'Gros Michel' bananas in the region. The isolates sampled from Ecuador caused disease in cultivars that are susceptible to races 1 and 2 under greenhouse experiments, although Fusarium wilt symptoms in the field were only found in 'Gros Michel'. Isolates belonging to the same VCG0120 have historically caused disease on Cavendish cultivars in the subtropics. Overall, this study shows how Foc can be easily dispersed to other areas if restriction of contaminated materials is not well enforced. We highlight the need of major efforts on awareness and monitoring campaigns to analyze suspected cases and to contain potential first introduction events of FocTR4 in Ecuador.
Project description:BACKGROUND:Banana Fusarium wilt is a devastating disease of bananas caused by Fusarium oxysporum f. sp. cubense (Foc) and is a serious threat to the global banana industry. Knowledge of the pathogenic molecular mechanism and interaction between the host and Foc is limited. RESULTS:In this study, we confirmed the changes of gene expression and pathways in the Cavendish banana variety 'Brazilian' during early infection with Foc1 and Foc4 by comparative transcriptomics analysis. 1862 and 226 differentially expressed genes (DEGs) were identified in 'Brazilian' roots at 48?h after inoculation with Foc1 and Foc4, respectively. After Foc1 infection, lignin and flavonoid synthesis pathways were enriched. Glucosinolates, alkaloid-like compounds and terpenoids were accumulated. Numerous hormonal- and receptor-like kinase (RLK) related genes were differentially expressed. However, after Foc4 infection, the changes in these pathways and gene expression were almost unaffected or weakly affected. Furthermore, the DEGs involved in biological stress-related pathways also significantly differed after infection within two Foc races. The DEGs participating in phenylpropanoid metabolism and cell wall modification were also differentially expressed. By measuring the expression patterns of genes associated with disease defense, we found that five genes that can cause hypersensitive cell death were up-regulated after Foc1 infection. Therefore, the immune responses of the plant may occur at this stage of infection. CONCLUSION:Results of this study contribute to the elucidation of the interaction between banana plants and Foc and to the development of measures to prevent banana Fusarium wilt.
Project description:Fusarium wilt caused by Fusarium oxysporum f.sp. cubense (Foc) is one of the most destructive diseases for banana. For their risk assessment and hazard characterization, it is vital to quickly determine the virulence of Foc isolates. However, this usually takes weeks or months using banana plant assays, which demands a better approach to speed up the process with reliable results. Foc produces various mycotoxins, such as fusaric acid (FSA), beauvericin (BEA), and enniatins (ENs) to facilitate their infection. In this study, we developed a linear regression model to predict Foc virulence using the production levels of the three mycotoxins. We collected data of 40 Foc isolates from 20 vegetative compatibility groups (VCGs), including their mycotoxin profiles (LC-MS) and their plant disease index (PDI) values on Pisang Awak plantlets in greenhouse. A linear regression model was trained from the collected data using FSA, BEA and ENs as predictor variables and PDI values as the response variable. Linearity test statistics showed this model meets all linearity assumptions. We used all data to predict PDI with high fitness of the model (coefficient of determination (R2 = 0.906) and adjust coefficient (R2adj = 0.898)) indicating a strong predictive power of the model. In summary, we developed a linear regression model useful for the prediction of Foc virulence on banana plants from the quantification of mycotoxins in Foc strains, which will facilitate quick determination of virulence in newly isolated Foc emerging Fusarium wilt of banana epidemics threatening banana plantations worldwide.