combined-recombinase-polymerase-amplification-crisprcas12a-assay-for

Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting <em>Fusarium oxysporum</em> f. sp. <em>cubense</em> Tropical Race 4

Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most ...

Stellenbosch University

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.

Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4.

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.

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

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/μL and a LOD of 10-4-10-5 ng/μL. 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.

Quantitative detection of economically important Fusarium oxysporum f. sp. cubense strains in Africa in plants, soil and water.

Fusarium wilt, caused by the soil-borne fungus  f. sp.  (Foc) race 1, is a major disease of bananas in East Africa. Triploid East African Highland (Matooke) bananas are resistant to Foc race 1, but the response of diploid (Mchare and Muraru) bananas to the fungus is largely unknown. A breeding project was initiated in 2014 to increase crop yield and improve disease and pest resistance of diploid and triploid East African Highland bananas. In this study, eight Mchare cultivars were evaluated for resistance to Foc race 1 in the field in Arusha, Tanzania. In addition, the same eight Mchare cultivars, as well as eight Muraru cultivars, 27 Mchare hybrids, 60 Matooke hybrids and 19 NARITA hybrids were also screened in pot trials. The diploid Mchare and Muraru cultivars were susceptible to Foc race 1, whereas the responses of Mchare, NARITAs and Matooke hybrids ranged from susceptible to resistant. The Mchare and Matooke hybrids resistant to Foc race 1 can potentially replace susceptible cultivars in production areas severely affected by the fungus. Some newly bred Matooke hybrids became susceptible following conventional breeding, suggesting that new hybrids need to be screened for resistance to all Foc variants.

Evaluation of Mchare and Matooke Bananas for Resistance to  f. sp.  Race 1.

Black Sigatoka, caused by , is a major foliar disease of banana and plantain worldwide. There are few available data regarding the genetic diversity and population structure of the pathogen in East Africa, which are needed to design effective and durable disease management strategies. We genotyped 319 single-spore isolates of .  collected from seven regions in Uganda and Tanzania and five isolates from Nigeria using 16 simple sequence repeat markers and mating type-specific primers. Isolates from each country and region within the country were treated as populations and subpopulations, respectively. A total of 296 multilocus genotypes (MLGs) were recovered, representing a clonal fraction of 7%. Subpopulations had a moderate level of genetic diversity (H = 0.12 to 0.31; mean, 0.29). Mating type distribution did not deviate from equilibrium (: , 1:1 ratio) in Uganda; however, in Tanzania the mating types were not in equilibrium (4:1 ratio). The index of association tests ( and ) showed that all populations were at linkage equilibrium ( > 0.05), thus supporting the hypothesis of random association of alleles. These findings are consistent with a pathogen that reproduces both clonally and sexually. Low and insignificant levels of population differentiation were detected, with 90% of the variation occurring among isolates within subpopulations. The high intrapopulation variation has implications in breeding for resistance to .  because isolates differing in aggressiveness and virulence are likely to exist over small spatial scales. Diverse isolates will be required for resistance screening to ensure selection of banana cultivars with durable resistance to Sigatoka in East Africa.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genetic Diversity and Mating Type Distribution of  on Banana in Uganda and Tanzania.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the  community voiced near unanimous support for a concept of  that represented a clade comprising all agriculturally and clinically important  species, including the  species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus  into seven genera, including the FSSC described as members of the genus , with subsequent justification in 2018 based on claims that the 2013 concept of  is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of  including the FSSC. We reassert the practical and scientific argument in support of a genus  that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus  were recombined in genus , and nine others were renamed  Here the global  community voices strong support for the inclusion of the FSSC in , as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic  that Includes the  Species Complex.

 f. sp  (Foc), the causal agent of Fusarium wilt, is one of the most devastating constraints to banana production worldwide. The spread of Foc in water is particularly concerning, as infested water can rapidly contaminate disease-free areas. The objectives of this study were to investigate the survival of Foc in water and to test the effectiveness of water treatment with chlorine, ozone, UV, and peracetic acid. The study indicated that Foc spores can survive in water for more than 120 days, but that viability was reduced in stagnant water, probably due to anaerobic conditions when spores settled at the bottom. It is therefore recommended that surface water be extracted and treated before it is used for irrigation. The efficacy of all water treatments was reduced in the presence of soil, implying that water needs to be soil-free before treatment. The use of peracetic acid is recommended to treat Foc-contaminated water, as it is safe for use and does not require installation costs although it is effective at treating Foc-contaminated water, ozone would require significant input costs and chlorine can produce harmful disinfection by-products. UV would be impractical for field application because of the high doses required to eliminate Foc.

The Survival and Treatment of  f. sp.  in Water.

Fusarium wilt, caused by the fungus  f. sp.  (Foc), poses a major threat to global banana production. The tropical race 4 (TR4) variant of Foc is a highly virulent form with a large host range, and severely affects Cavendish bananas. Foc TR4 was recently observed within the Greater Mekong Subregion, after Chinese private companies expanded Cavendish production to the region. In this study, extensive surveys conducted across Laos and Vietnam show that Foc TR4 is still mainly constricted to the northern regions of these countries and is limited to Cavendish cultivation settings. In Laos, Foc TR4 is associated with large-scale Cavendish plantations owned by or involved with Chinese companies through which infected planting material could have been imported. In Vietnam, mostly small-holder Cavendish farmers and backyard gardens were affected by Foc TR4. In Vietnam, no direct link is found with Chinese growers, and it is expected the pathogen mainly spreads through local and regional movement of infected planting materials. Foc TR4 was not recorded on banana cultivars other than Cavendish. The extensively cultivated 'Pisang Awak' cultivar was solely infected by VCGs belonging to Foc race 1 and 2, with a high occurrence of VCG 0123 across Laos, and of VCG 0124/5 in Vietnam. Substantial diversity of Foc VCGs was recorded (VCGs 0123, 0124/5, 01218 and 01221) from northern to southern regions in both countries, suggesting that Fusarium wilt is well established in the region. Interviews with farmers indicated that the local knowledge of Fusarium wilt epidemiology and options for disease management was limited. Clear communication efforts on disease epidemiology and management with emphasis on biosecurity practices need to be improved in order to prevent further spread of Foc TR4 to mixed variety smallholder settings.

Geographical Distribution and Genetic Diversity of the Banana Fusarium Wilt Fungus in Laos and Vietnam.

 f. sp.  (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  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  () 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  genes mostly conserved within these clusters. Results from this study indicates that Foc is more diverse than hitherto assumed.

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

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to  f. sp.  (), the causal agent of this disease, is genetically dissected in this study using two  ssp.  segregating populations, segregating for  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 F 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.

Genetic Mapping, Candidate Gene Identification and Marker Validation for Host Plant Resistance to the Race 4 of  f. sp.  Using  ssp. .

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most damaging plant diseases known. Foc race 1 (R1) decimated the Gros Michel-based banana (Musa acuminata) trade, and now Foc tropical race 4 (TR4) threatens global production of its replacement, the Cavendish banana. Here population genomics revealed that all Cavendish banana-infecting Foc race 4 strains share an evolutionary origin distinct from that of R1 strains. Although TR4 lacks accessory chromosomes, it contains accessory genes at the ends of some core chromosomes that are enriched for virulence and mitochondria-related functions. Meta-transcriptomics revealed the unique induction of the entire mitochondrion-localized nitric oxide (NO) biosynthesis pathway upon TR4 infection. Empirically, we confirmed the unique induction of a NO burst in TR4, suggesting that nitrosative pressure may contribute to virulence. Targeted mutagenesis demonstrated the functional importance of fungal NO production and the accessory gene SIX4 as virulence factors.

Virulence of banana wilt-causing fungal pathogen Fusarium oxysporum tropical race 4 is mediated by nitric oxide biosynthesis and accessory genes.

The soilborne pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is currently devastating banana production worldwide. Once introduced, it is not possible to eradicate the pathogen from soils where it can survive for decades. The only management option available then is to replace Foc TR4-susceptible with -resistant varieties. Timely detection of the pathogen, however, is an important strategy to prevent the introduction of Foc TR4 into new areas and prevent its spread from infested sites. In this study, a single-tube detection technique was developed by combining recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology (RPA-Cas12a) for detection of Foc TR4. The RPA-Cas12a assay was conducted isothermally, had a sensitivity of up to 10 fg target DNA and did not cross react with any of the 76 non-target isolates included in the specificity testing. The RPA-Cas12a assay detected Foc TR4 from naturally infected banana samples collected in the field and visualization was possible with the naked eye under LED blue light transillumination. The method can be integrated with inexpensive fluorescent or electronic detection devices to accelerate Foc TR4 in-field detection and, thereby, fast-track disease containment strategies.

A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4.

Leptosphaeria maculans is the causal agent of blackleg, a globally important disease of canola. Investigating the genetic diversity and structure of L. maculans populations can provide insight into its evolutionary potential and genetic variability, which is important to develop effective blackleg management strategies. In this study, whole genome sequence data was generated for 230 L. maculans isolates collected between 2020 and 2022 across the canola production regions of the Western Cape of South Africa. A total of 27 419 informative single nucleotide polymorphisms was used to investigate the genetic diversity and structure of the pathogen population.

Whole genome sequencing reveals the genetic diversity and structure of Leptosphaeria maculans populations from the Western Cape province of South Africa.

Author Correction: Virulence of banana wilt-causing fungal pathogen Fusarium oxysporum tropical race 4 is mediated by nitric oxide biosynthesis and accessory genes.

Fusarium wilt (Foc) TR4 was first reported in Northern Vietnam in 2018. Since then, it has rapidly spread across most northern provinces along the Red River basin banana production landscapes, impacting Cavendish ( AAA genome) production. The other main banana cultivars which are widely grown in this production zone are Pisang Awak ( ABB genome) and Pisang Mas ( AA genome). Field surveys were conducted in 2022/2023 across this banana production region to assess pathogen spread from Cavendish monocropping systems into adjacent smaller-scale mixed cultivar systems. Across 130 sites, a total of 210 banana pseudostem tissue samples were collected from symptomatic Cavendish, Pisang Awak and Pisang Mas plants. Foc TR4 incursions into mixed small-to-mid-sized Cavendish-Pisang Awak plantations were confirmed, and the pathogen was also recorded in Pisang Awak plantations and backyard gardens that did not contain any Cavendish mats. A screenhouse-based Foc TR4 screening trial including seven commonly cultivated  varieties in Northern Vietnam indicated that Pisang Awak and Pisang Mas are susceptible to the pathogen. While Pisang Awak, an important local variety, is known to be susceptible to both Foc Race 1 and TR4, recent field observations suggest a limited susceptibility of Pisang Awak to Foc TR4 in mixed cultivar plantation settings. Local farmers similarly reported observing reduced susceptibility, with several having already replanted TR4-affected Cavendish fields with Pisang Awak as part of their disease management strategy. No infections were observed on field-grown Pisang Mas plants in TR4-affected mixed banana cultivar production landscapes. These results and insights provide solutions for the revival of TR4-affected Cavendish production fields or landscapes, through the cultivation of less susceptible local cultivars. In addition, the introduction, validation and scaling of Formosana (i.e., GCTCV-218, a Cavendish somaclone with moderate resistance to Foc TR4) should be envisaged.

Article	Year
Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4. PloS one\| PubMed ID: 28178348	2017
The distribution and host range of the banana Fusarium wilt fungus, Fusarium oxysporum f. sp. cubense, in Asia. PloS one\| PubMed ID: 28719631	2017
Quantitative detection of economically important Fusarium oxysporum f. sp. cubense strains in Africa in plants, soil and water. PloS one\| PubMed ID: 32687514	2020
Evaluation of Mchare and Matooke Bananas for Resistance to f. sp. Race 1. Plants (Basel, Switzerland)\| PubMed ID: 32842551	2020
Genetic Diversity and Mating Type Distribution of on Banana in Uganda and Tanzania. Phytopathology\| PubMed ID: 32931393	2021
Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic that Includes the Species Complex. Phytopathology\| PubMed ID: 33200960	2021
The Survival and Treatment of f. sp. in Water. Journal of fungi (Basel, Switzerland)\| PubMed ID: 34682219	2021
Geographical Distribution and Genetic Diversity of the Banana Fusarium Wilt Fungus in Laos and Vietnam. Journal of fungi (Basel, Switzerland)\| PubMed ID: 35049986	2022
A Polyphasic Approach Reveals Novel Genotypes and Updates the Genetic Structure of the Banana Fusarium Wilt Pathogen. Microorganisms\| PubMed ID: 35208723	2022
Genetic Mapping, Candidate Gene Identification and Marker Validation for Host Plant Resistance to the Race 4 of f. sp. Using ssp. . Pathogens (Basel, Switzerland)\| PubMed ID: 37375510	2023
Virulence of banana wilt-causing fungal pathogen Fusarium oxysporum tropical race 4 is mediated by nitric oxide biosynthesis and accessory genes. Nature microbiology\| PubMed ID: 39152292	2024
A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4. Scientific reports\| PubMed ID: 39828694	2025
Whole genome sequencing reveals the genetic diversity and structure of Leptosphaeria maculans populations from the Western Cape province of South Africa. BMC genomics\| PubMed ID: 40181296	2025
Author Correction: Virulence of banana wilt-causing fungal pathogen Fusarium oxysporum tropical race 4 is mediated by nitric oxide biosynthesis and accessory genes. Nature microbiology\| PubMed ID: 40987852	2025
The Evolving Threat of Fusarium Wilt TR4 to Small-Scale Mixed Cultivar Banana Production in the Red River Basin of Northern Vietnam. Journal of fungi (Basel, Switzerland)\| PubMed ID: 41003199	2025

Diane Mostert

Biography

JoVE Journal Publications

Other Publications