A Detection Method for Tropical Race 4 of the Banana Pathogen Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Fusarium wilt, the devastating disease that ruined the ‘Gros Michel’ (AAA)-based banana production in the first half of the 20th century. The occurrence of a new variant in Southeast Asia that overcomes the resistance in Cavendish clones such as ‘Grand Naine’ (AAA) is a major concern to current banana production worldwide. The threat posed by this new variant, called tropical race 4 (TR4), may be overcome by the introduction of resistant cultivars. However, the identification of new resistant sources or breeding for resistance is a long-term effort. Currently, the only option to control the disease is to avoid or reduce the spread of the pathogen by eradication of infected plants and isolation of infested plantations. This requires sensitive and highly specific diagnostics that enable early detection of the pathogen. A two-locus database of DNA sequences, from over 800 different isolates from multiple formae speciales of F. oxysporum, was used to develop a molecular diagnostic tool that specifically detects isolates from the vegetative compatibility group (VCG) 01213, which encompasses the Foc TR4 genotype. This diagnostic tool was able to detect all Foc TR4 isolates tested, while none of the Foc isolates from 19 VCGs other than 01213 showed any reaction. In addition, the developed diagnostic tool was able to detect Foc TR4 when using DNA samples from different tissues of ‘Grand Naine’ plants inoculated with TR4 isolate

The distribution and host range of the banana Fusarium wilt fungus, Fusarium oxysporum f. sp. cubense, in Asia

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.S1 Table. Morphological identity, PCR-RFLP clade and lineage identity, vegetative compatibility group, host and host subgroup, location and origin of Fusariumisolates collected in Asia.S2 Table. Vegetative compatibility tester strains used to characterize compatibility to Asian Fusariumoxysporumf. sp. cubense isolates.http://www.plosone.orgam2018Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

A Polyphasic Approach Reveals Novel Genotypes and Updates the Genetic Structure of the Banana Fusarium Wilt Pathogen

Fusarium oxysporum f. sp. cubense (Foc) is a soil-borne fungus that causes Fusarium wilt, a destructive plant disease that has resulted in devastating economic losses to banana production worldwide. The fungus has a complex evolutionary history and taxonomic repute and consists of three pathogenic races and at least 24 vegetative compatibility groups (VCGs). Surveys conducted in Asia, Africa, the Sultanate of Oman and Mauritius encountered isolates of F. oxysporum pathogenic to banana that were not compatible to any of the known Foc VCGs. Genetic relatedness between the undescribed and known Foc VCGs were determined using a multi-gene phylogeny and diversity array technology (DArT) sequencing. The presence of putative effector genes, the secreted in xylem (SIX) genes, were also determined. Fourteen novel Foc VCGs and 17 single-member VCGs were identified. The multi-gene tree was congruent with the DArT-seq phylogeny and divided the novel VCGs into three clades. Clustering analysis of the DArT-seq data supported the separation of Foc isolates into eight distinct clusters, with the suite of SIX genes mostly conserved within these clusters. Results from this study indicates that Foc is more diverse than hitherto assumed

Uji Virulensi Isolat Fusarium Oxysporum F.sp. Cubense Dalam Vegetative Compatibility Group Complex 0124 Pada Tanaman Pisang

Patogen Fusarium oxysporum f.sp. cubense (Foc) ras 1 dilaporkan tidak patogenik terhadap pisang kelompok Cavendish, namun beberapa hasil penelitian menunjukkan bahwa ras 1 pada kondisi tertentu juga dapat menyerang pisang kelompok Cavendish. Sedikit laporan tentang variasi virulensi Foc ras 1 dalam VCG complex 0124 terhadap pisang Ambon Kuning (AAA/Gros Michel) dan Ambon Hijau (AAA/Cav.subgroup). Tujuan penelitian ialah mengetahui variasi virulensi isolat Foc dalam VCG complex 0124 terhadap pisang Ambon Kuning dan Ambon Hijau. Penelitian dilakukan di Laboratorium Proteksi dan Rumah Kasa Balai Penelitian Tanaman Buah Tropika Solok, dari Bulan April sampai Oktober 2011. Rancangan yang digunakan ialah acak kelompok faktorial dengan tiga ulangan setiap perlakuan terdiri atas 10 tanaman. Faktor pertama ialah lima isolat Foc dalam VCG complex 0124: F1=0124/5 (WJP 02), F2=0124/5/8 (WJG 03), F3=0124/5/8 (WJG 09), F4=0124/5 (Indo 119), dan F5=0124/5/20 (02020114B) dan faktor kedua ialah dua varietas pisang yaitu: V1= varietas Ambon Hijau (AAA/Cav.subgroup) dan V2=varietas Ambon Kuning (AAA/Gros Michel). Hasil penelitian menunjukkan bahwa semua isolat Foc dalam VCG complex 0124 yang diuji dapat menyerang pisang Ambon Kuning dan Ambon Hijau dengan persentase serangan yang sangat tinggi yaitu 96,67–98,33%. Perlakuan tunggal jenis isolat terhadap peubah masa inkubasi menunjukkan bahwa isolat 0124/5/8 (WJG 03) masa inkubasinya paling panjang yaitu 26,57 hari dan berbeda nyata dengan isolat lainnya. Masa inkubasi penyakit oleh semua isolat yang diuji pada Ambon Kuning lebih panjang (24,27 hari) dan berbeda nyata dengan Ambon Hijau (16,42 hari). Semua isolat Foc yang diuji sangat virulen pada pisang Ambon Kuning (AAA/Gros Michel) dan Ambon Hijau (AAA/Cav.subgroup) dengan indeks keparahan penyakit pada daun dan bonggol berkisar antara 4,72–5,22 dan 5,03–5,14. Hasil penelitian ini mendorong kajian lebih lanjut tentang biologi dan virulensi patogen dalam rangka memperoleh teknik pengendalian yang tepat

Characterization Of Populations Of Fusarium Oxysporum F. Sp. Cubense And Selection Of Somaclonal Variants Tolerant To Banana Wilt Desease

Sebanyak 40 isolat Fusarium oxysporum f. sp. cubense (FOC) dari Indonesia, Sumatra, dan Semenanjung Malaysia yang dipencil dan pelbagai variety pisang utama di Asia tenggara telah dikenalpasti dari segi morfologi, ras dan keserasian vegetatifnya. Keputusan analisis keserasian vegetatif (VCG) menunjukkan kehadiran 'clonal lineages' di Semenanjung Malaysia. Sebanyak 12 kelompok keserasian vegetatif telah dikenalpasti. Lima kelompok adalah sarna dengan VCG 0121, VCG 0124, VCG 01213, VCG 01216 dan VCG 01213/01216. Tujuh kelompok yang lain tidak dapat dikategorikan menggunakan sistem VCG yang sedia ada. Forty isolates of Fusarium oxysporum f. sp cubense (Foc) located in Indonesia (Sumatra) and Peninsular Malaysia were isolated from a diverse banana variety host range which included all significant South East Asian cultivars were characterized by morphology, race and vegetative compatibility (VCG). There appeared no direct pattern of association between race, VCG and morphological characteristics. VCG analysis indicated that there were several distinct clonal lineages present in Peninsular Malaysia. Twelve compatibility groups were identified, five of which were previously characterized as VCG 0121, VCG 0124, VCG 01213, VCG 01216 and VCG 01213/01216

Phylogenetic analysis of Fusarium oxysporum f. sp. cubense associated with fusarium wilt of bananas from Peninsular Malaysia

Fusarium wilt of bananas caused by Fusarium oxysporum f. sp. cubense (Foc) has been known to hamper banana production. In Malaysia, most of the edible banana cultivars are susceptible to the fungal strain Foc tropical race 4 (Foc TR4). To date, molecular characterisation of Foc TR4 in this country remains elusive. Thus, this study aimed to characterise the diversity of Foc TR4 in Peninsular Malaysia based on the phylogenetic analysis of transcription elongation factor (TEF-1α) sequences. From March 2016 to December 2016, 17 isolates of Foc were isolated from nine outbreak states in Peninsular Malaysia consisting of Penang, Perak, Selangor, Negeri Sembilan, Melaka, Johor, Pahang, Kelantan and Terengganu, with disease incidence exceeding 70% in several plantations. Morphological, molecular identification using TR4 specific primers and pathogenicity assay confirmed the identity of the fungal pathogen. Pathogenicity assays indicated that the aggressiveness level varies among the isolates. Phylogenetic analyses of TEF1-α sequences showed that there was no genetic variation among the Foc isolates by clustering them into four vegetative compatibility groups (VCGs) of 01213 (TR4), 01216 (TR4), 01213/16 (TR4) and 0121 (R4). PCR amplification using VCG specific primers have further categorised 16 Foc isolates as VCG01213 and a single isolate as VCG01213/16. In addition, no correlation between TEF-1α sequences and aggressiveness of isolates could be established

Molecular diagnostics of banana Fusarium wilt targeting Secreted-in-Xylem genes

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 variety succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial variety worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to Cavendish and other commonly grown varieties. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana varieties. 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, TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas

Fusarium wilt of banana: Global problems and perspectives

Fusarium wilt of banana is recognized as one of the most destructive diseases of banana worldwide. In addition to an overview of the history of research into fusarium wilt of banana, a precis of the current global problems posed by this disease to producers and consumers of bananas is presented in this paper. Key issues and opportunities facing scientific researchers in their attempts to find solutions to the management of this disease are also discussed, with reference to the notion of sustainable agriculture

Evaluation of Some Specific Primer Sets Development for Detecting Fusarium oxysporum f. sp. cubense Tropic Race 4 (Foc TR4) Originating from Indonesia

Fusarium oxysporum f. sp. cubense tropic race 4 (Foc TR4) strain which belong to Vegetative Compatibility Group (VCG) 01213 is the most devastating disease in global banana production. Validation of specific primer sets using the positive control (Foc TR4). In total, 50 isolates of Foc are collected from several banana production regions in Indonesia represent the group of VCG, races, genotype, cultivars, which are confirmed as Foc based on the tested using FocEf3 primer set, except Cjr-2 and Lmp-4 isolates. Foc-1/Foc-2 could amplify 34 Foc isolates included in Foc race 4. Three specific primer sets i.e. TR4-F/TR4-R, Six-1c, and TR4-F2/TR4-R1 are used to classify Foc isolates into Foc tropic race 4. TR4-F/TR4-R is known have the highest specificity as it could amplify 35 Foc isolates including positive controls (Foc TR4) compared to the other primer sets (Six-1c and TR4-F2/TR4-R1). This research indicates that there are a large number of diversity strains found in Foc isolates to be studied for further research. Race 4 of Foc (STR4 or TR4) is known to be widespread in several regions in Indonesia. Therefore, specific primer set development needs to be done to detect Foc TR4 and the most damaging strains on Foc TR4 based on molecular data. Intisari Fusarium oxysporum f. sp. cubense ras 4 tropika (Foc TR4) yang termasuk ke dalam kelompok VCG 01213 merupakan patogen yang paling merusak dalam produksi tanaman pisang secara global. Validasi primer spesifik berbasis PCR menggunakan kontrol positif (Foc TR4). Total, 50 isolat Foc dikoleksi dari  beberapa daerah produksi pisang di Indonesia mewakili VCG, ras, genotipe dan kultivar yang dikonfirmasi sebagai isolat Foc berdasarkan pengujian menggunakan primer FocEf3, kecuali isolat Cjr-2 dan Lmp-4. Foc-1/Foc-2 dapat mengamplifikasi 34 isolat Foc yang termasuk ke dalam Foc ras 4. Selanjutnya tiga pasang primer spesifik yaitu TR4-F/TR4-R, Six-1c, dan TR4-F2/TR4-R1 digunakan untuk mengelompokkan isolat-isolat tersebut ke dalam isolat Foc ras 4 tropika. TR4-F/TR4-R diketahui memiliki spesifitas tertinggi karena dapat mengamplifikasi sebanyak 35 isolat Foc termasuk kontrol positif (Foc TR4) dibandingkan dengan primer lainnya (Six-1c dan TR4-F2/TR4-R1). Penelitian ini menunjukkan bahwa terdapat sejumlah besar keragaman strain yang terlihat pada isolat-isolat Foc tersebut untuk dapat dipelajari lebih lanjut. Ras 4 dari Foc (STR4 atau TR4) diketahui tersebar luas pada beberapa daerah di Indonesia. Oleh karena itu, perlu dilakukan pengembangan primer spesifik untuk mendeteksi Foc TR4 dan strain yang paling merusak pada Foc TR4 berdasarkan data molekuler