Accessory genes in tropical race 4 contributed to the recent resurgence of the devastating disease of Fusarium wilt of banana.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most damaging plant diseases recorded. Foc race 1 (R1) decimated the Gros Michel-based banana trade. Currently, tropical race 4 (TR4) is threatening the global production of its replacement cultivar, Cavendish banana. Population genomics and phylogenetics revealed that all Cavendish banana-infecting race 4 strains shared an evolutionary origin that is distinct from R1 strains. The TR4 genome lacks accessory or pathogenicity chromosomes, reported in other F. oxysporum genomes. Accessory genes-enriched for virulence and mitochondrial-related functions-are attached to ends of some core chromosomes. Meta-transcriptomics revealed the unique induction of the entire mitochondria-localized nitric oxide (NO) biosynthesis pathway upon TR4 infection. Empirically, we confirmed the unique induction of NO burst in TR4,suggesting the involvement of nitrosative pressure in its virulence. Targeted mutagenesis demonstrated the functional importance of accessory genes SIX1 and SIX4 as virulent factors.

First report of Fusarium wilt of Cavendish bananas caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 in the Grande Comoros Island.

Fusarium wilt of banana, caused by the soil-borne pathogen Fusarium oxysporum f. sp. cubense (Foc), is a major constraint to banana production worldwide (Viljoen et al., 2020). Currently, Cavendish bananas are severely affected by Foc Tropical Race 4 (TR4) globally. In Africa, Foc TR4 was first detected in northern Mozambique in 2013 (Viljoen et al., 2020), and has since been found on the island of Mayotte in the Mozambique Channel off the coast of southeastern Africa (Aguayo et al., 2021). In early 2023, severe leaf-yellowing and wilting of Cavendish banana plants was observed on Cavendish banana plants at several small holder farmer properties in Grande Comoros (Ngazidja) including in Ntsinimoipanga (-11,790054 S, 43°25'47,04384 E), Batou (-11,499716 S, 43°21'51,71976''E), Madjeweni (-11,8217 S , 43°16'41''E) and Mdé (-11°41'54'' S, 43°15'20''E). When the pseudostems of these plants were split open, a reddish-brown internal discoloration of the vascular tissue became apparent. Discolored strands of diseased plants were collected, and the causal agent identified using DNA-based techniques, vegetative compatibility group (VCG) analysis and pathogenicity testing. The samples were plated onto potato dextrose agar and single-spored and isolated from individual diseased plants identified as F. oxysporum based on cultural and morphological characteristics. These include the production of white fungal colonies with a purple center, infrequent production of macroconidia, but an abundance of microconidia on short monophialides, and terminal or intercalary chlamydospores (Leslie and Summerell, 2006). Foc TR4 was identified from seven isolates by conventional (Dita et al., 2010) and quantitative-PCR (Matthews et al., 2020), and with loop-mediated isothermal amplification (LAMP) (Ordóñez et al., 2021). All seven isolates were confirmed as members of the VCG 01213/16 complex when nit-1 mutants of the unknown Foc isolates were compatible with Nit-M mutants of the Foc VCG 01213 and VCG 01216 tester strains. Two isolates were then selected for pathogenicity testing, and 2-month-old tissue culture-derived Cavendish plants (cv. Williams) inoculated by using the method described by Ndayihanzamaso et al. (2022). After 4 weeks, the Foc TR4-inoculated plants produced wilting symptoms and internal rhizome discoloration typical of Fusarium wilt. Foc TR4 was reisolated from the inoculated plants and identified by qPCR (Matthews et al. 2020), thereby fulfilling Koch's postulates. These results provide scientific proof of the presence of Foc TR4 in a second island in the Comoros archipelago. Comprehensive surveys will be conducted in all three of the Comoros Islands to assess the presence and impact of Foc TR4 to implement containment strategies. Collaborative initiatives and coordinated actions among growers and other stakeholders are needed to prevent the spread of Foc TR4 to more Southwest Indian Ocean islands and countries on the East African coasts. The importance of banana for food security and livelihoods, and the unique genetic diversity of bananas found on the Comoros islands, requires the eradication and isolation of diseased bananas on the short term, and the screening of local banana varieties for Foc TR4 resistance on the longer term.

First report of Fusarium wilt of Cavendish bananas caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 in Venezuela.

Fusarium wilt of banana (Musa spp.), caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is a major constraint to banana production worldwide (Dita et al., 2018). A strain of Foc that affects Cavendish (AAA) bananas in the tropics, called Foc tropical race 4 (TR4; VCG 01213), is of particular concern. Foc TR4 was first detected in Malaysia and Indonesia around 1990 but was restricted to Southeast Asia and northern Australia until 2012. The fungus has since been reported from Africa, the Indian subcontinent, and the Middle East (Viljoen et al., 2020). Foc TR4 was detected in Colombia in 2019 and in Perú in 2021 (Reyes-Herrera et al., 2020). The incursions into Latin America and the Caribbean (LAC) triggered global concerns, as 75% of international export bananas are produced in the region. Banana production in Venezuela, however, is primarily intended for domestic consumption (Aular and Casares, 2011). In 2021 the country produced 533,190 metric tons of banana on an area of 35,896 ha, with an approximate yield of 14,853 kg/ha (FAOSTAT, 2023). In July 2022, severe leaf-yellowing, and wilting, along with internal vascular discoloration of the pseudostem, were noted in Cavendish banana plants cultivar 'Valery' in the states of Aragua (10°11'8″N; 67°34'51″W), Carabobo (10º14'24″N; 67º48'51″W), and Cojedes (9°37'44″N; 68°55'4″W). Necrotic strands from the pseudostems of diseased plants were collected for identification of the causal agent using DNA-based techniques, vegetative compatibility group (VCG) analysis and pathogenicity testing. The samples were first surface disinfected and plated onto potato dextrose agar medium. Single-spored isolates were identified as F. oxysporum based on cultural and morphological characteristics, including white colonies with purple centres, infrequent macroconidia, abundant microconidia on short monophialides, and terminal or intercalary chlamydospores (Leslie and Summerell, 2006). Foc TR4 was identified from five isolates by endpoint and quantitative-PCR using four different primer sets (Li et al. 2013; Dita et al. 2010; Aguayo et al. 2017; Matthews et al. 2020). The same isolates were identified as VCG 01213 by successfully pairing nitrate non-utilizing (nit-1) mutants of the unknown strains with Nit-M testers of Foc TR4 available at Stellenbosch University (Leslie and Summerell, 2006). For pathogenicity testing, 3-month-old Cavendish banana plants cultivar 'Williams' were inoculated with isolates from Venezuela grown on sterile millet seed (Viljoen et al., 2017). Plants developed typical Fusarium wilt symptoms 60 days after inoculation, including yellowing of leaves that progressed from the older to the younger leaves, wilting, and internal discoloration of the pseudostem. Koch's postulates were fulfilled by reisolating and identifying Foc TR4 from the plants by qPCR (Matthews et al., 2020). These results provide scientific proof of the presence of Foc TR4 in Venezuela. The Venezuelan Plant Protection Organization (INSAI) has declared Foc TR4 as a newly introduced pest (January 19, 2023), and infested banana fields were placed under quarantine. Comprehensive surveys are now conducted in all production areas in Venezuela to assess the presence and impact of Foc TR4, and information campaigns were started to make farmers aware of biosecurity protocols. Collaborative initiatives and coordinated actions among all stakeholders are needed to prevent the spread of Foc TR4 to other countries in Latin America, and to develop Foc TR4-resistant bananas (Figueiredo et al. 2023).

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.

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.

FocECM33, a GPI-anchored protein, regulates vegetative growth and virulence in Fusarium oxysporum f. sp. cubense tropical race 4.

ECM33, a glycosylphosphatidylinositol (GPI)-anchored protein, is important for fungal development and infection through regulating fungal cell wall integrity, however, the functions of its orthologs in pathogenesis have not been characterized in Fusarium oxysporum. Here, we discovered a GPI-anchored protein, FocECM33, which is required for vegetative growth and virulence of Fusasium oxysporum f. sp. cubense tropical race 4 (Foc TR4). FocECM33 was highly upregulated during the early infection process of Foc TR4 in banana roots. The targeted disruption of FocECM33 led to decreased hyphal growth, increased sensitivity to cell wall stresses and reduced virulence on banana plantlets. Furthermore, ΔFocECM33 mutant demonstrated a cell morphology defect, elevated ROS production and increased chitin content. Transcriptome analysis showed that FocECM33 has a significant influence on the production of various secondary metabolites and regulation of many biosynthetic processes in Foc TR4. Taken together, it seems FocECM33 contributes to the virulence of Foc TR4 through regulating the process of hyphal growth, ROS production and chitin synthesis.

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

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. cubense (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.

The Survival and Treatment of Fusarium oxysporum f. sp. cubense in Water.

Fusarium oxysporum f. sp cubense (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 M35 Metalloprotease Effector FocM35_1 Is Required for Full Virulence of Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana, the most devastating disease on a banana plant. The genome of Foc TR4 encodes many candidate effector proteins. However, little is known about the functions of these effector proteins on their contributions to disease development and Foc TR4 virulence. Here, we discovered a secreted metalloprotease, FocM35_1, which is an essential virulence effector of Foc TR4. FocM35_1 was highly upregulated during the early stages of Foc TR4 infection progress in bananas. The FocM35_1 knockout mutant compromised the virulence of Foc TR4. FocM35_1 could interact with the banana chitinase MaChiA, and it decreased banana chitinase activity. FocM35_1 induced cell death in Nicotiana benthamiana while suppressing the INF1-induced hypersensitive response (HR), and its predicted enzymatic site was required for lesion formation and the suppression to INF1-induced HR on N. benthamiana leaves. Importantly, treatment of banana leaves with recombinant FocM35_1 accelerates Foc TR4 infection. Collectively, our study provides evidence that metalloprotease effector FocM35 seems to contribute to pathogen virulence by inhibiting the host immunity.

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

Black Sigatoka, caused by Pseudocercospora fijiensis, 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 P. fijiensis 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 (Hexp = 0.12 to 0.31; mean, 0.29). Mating type distribution did not deviate from equilibrium (MAT1-1: MAT1-2, 1:1 ratio) in Uganda; however, in Tanzania the mating types were not in equilibrium (4:1 ratio). The index of association tests (IA and r̄d) showed that all populations were at linkage equilibrium (P > 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 P. fijiensis 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.

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

Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (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.

First report of Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) causing banana wilt in the Island of Mayotte.

Fusarium oxysporum f. sp. cubense (Foc) is a fungus causing Fusarium wilt of banana (Musa spp.). The fungus is divided into three races and 24 vegetative compatibility groups (VCG) of which VCG 01213/16, commonly known as Foc tropical race 4 (Foc TR4), is of particular concern. Foc TR4 severely affects Cavendish (AAA) bananas, which comprise about 50% of all bananas produced globally, as well as many varieties susceptible to the other races of Foc. The pathogen was restricted to Southeast Asia and Australia until 2012, where after it has been detected in the Middle East, Mozambique in Africa, and Colombia in South America (Viljoen et al. 2020). Here we report the first detection of Foc TR4 in the French department of Mayotte, located in the Indian Ocean. In September 2019, leaf yellowing and wilting symptoms were observed in individual plants of the banana subgroups Silk (AAB) (cv. "Kissoukari") and Bluggoe (ABB) (cv. "Baraboufaka"). The symptomatic individuals were found in private gardens in the village of Poroani in Southwest Mayotte (World Geodetic System [WGS] 12° 53' 31.83''S, 45° 8' 30.98" E). When the pseudostems of symptomatic plants were split open, dark red to brown vascular discoloration was observed. Pseudostem tissue samples were collected and identified as Foc TR4 with the real-time PCR assay developed by Aguayo et al. (2017). Sections of the pseudostem samples were surface sterilized and used to isolate the fungus on potato dextrose agar (PDA) medium. Isolates were identified as F. oxysporum based on cultural and morphological characteristics as described in Leslie and Summerell (2006), which included fluffy aerial mycelia on PDA and the presence of short monophialides conidigenous cells bearing microconidia arranged in false heads. Abundant chlamydospores were also produced on synthetic nutrient poor agar (SNA) media. Single-spored isolates were used to develop nit mutants for vegetative compatibility group (VCG) testing (Correll 1991; Puhalla 1985). The isolates were confirmed as VCG 01213/16 as formation of heterokaryons was obtained with the nit mutants of the universal Foc TR4 tester. Two VCG 01213/16 isolates were then selected for pathogenicity testing by inoculating 2-month-old tissue culture-derived Cavendish plants, using the method described by Viljoen et al. (2017). After 10 weeks, the Foc TR4-inoculated plants produced wilting symptoms and internal rhizome discoloration typical of Fusarium wilt. Fusarium oxysporum was re-isolated from the inoculated plants and identified as Foc TR4/VCG 01213/16 by PCR (Dita et al. 2010; Matthews et al. 2020), thereby fulfilling Koch's postulates. Local authorities have destroyed the infected plants, and have undertaken an extensive survey to determine the distribution of Foc TR4 on the island. Three additional positive cases, identified with the real-time PCR assay of Aguayo et al. (2017), were found in the localities of Koungou ([WGS] 12° 44' 03''S, 45° 12' 08" E) and Bouéni ([WGS] 12° 54' 25''S, 45° 04' 43" E). These included infected Cavendish banana (AAA) plants (cv. "Kontriké"). This is the first time that Foc TR4 has been found on a banana variety other than Cavendish when newly detected in a country. Considering the proximity of Mayotte to other islands of the Comoros archipelago, Madagascar and the East African coast, where banana is considered an important staple, this report describes a serious threat to banana production and the livelihoods of people in the region.

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

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.

Fusaric acid instigates the invasion of banana by Fusarium oxysporum f. sp. cubense TR4.

Fusaric acid (FSA) is a phytotoxin produced by several Fusarium species and has been associated with plant disease development, although its role is still not well understood. Mutation of key genes in the FSA biosynthetic gene (FUB) cluster in Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) reduced the FSA production, and resulted in decreased disease symptoms and reduced fungal biomass in the host banana plants. When pretreated with FSA, both banana leaves and pseudostems exhibited increased sensitivity to Foc TR4 invasion. Banana embryogenic cell suspensions (ECSs) treated with FSA exhibited a lower rate of O2 uptake, loss of mitochondrial membrane potential, increased reactive oxygen species (ROS) accumulation, and greater nuclear condensation and cell death. Consistently, transcriptomic analysis of FSA-treated ECSs showed that FSA may induce plant cell death through regulating the expression of genes involved in mitochondrial functions. The results herein demonstrated that the FSA from Foc TR4 functions as a positive virulence factor and acts at the early stage of the disease development before the appearance of the fungal hyphae in the infected tissues.

Genetic Diversity of Fusarium oxysporum f. sp. cubense in East and Central Africa.

Banana Fusarium wilt is a major production constraint globally and a significant threat to the livelihoods of millions of people in East and Central Africa (ECA). A proper understanding of the diversity and population dynamics of the causal agent, Fusarium oxysporum f. sp. cubense (Foc), could be useful for the development of sustainable disease management strategies for the pathogen. The current study investigated the diversity of Foc in ECA using vegetative compatibility group (VCG) analysis, PCR-RFLPs of the ribosomal DNA's intergenic spacer region, as well as phylogenetic analysis of the elongation factor-1α gene. Six VCGs (0124, 0125, 0128, 01212, 01220, and 01222), which all belong to one lineage (Foc lineage VI), were widely distributed throughout the region. VCGs 0128 and 01220 are reported for the first time in Burundi, the Democratic Republic of Congo (DRC), Rwanda, Tanzania, and Uganda, while VCG 01212 is reported in the DRC and Rwanda. Isolates that did not belong to any of the known VCGs were identified as Foc lineage VI members by phylogenetic analysis and may represent novel VCGs. CAV 2734, a banana pathogen collected in Rwanda, clustered with nonpathogenic F. oxysporum isolates in lineage VIII. Results from this study will contribute significantly toward the implementation of banana Fusarium wilt disease management practices in the region, such as the restricted movement of infected planting material and the selective planting of resistant banana varieties.

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.

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 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.