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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 ‘DHPahang’ 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. ispartof: Pathogens vol:12 issue:6 pages:820- ispartof: location:Switzerland status: Published online

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.

Banana cultivars with the AAB genome group comprise diverse subgroups, such as Plantain, Silk, Iholena, and Pisang Raja, among others, which play an important role in food security in many developing countries. Some of these cultivars are susceptible to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), the most destructive pathogen threatening banana production worldwide, and some of them are still largely unknown. We evaluated the resistance of 37 banana genotypes, including Plantain, Silk, Iholena, Maia Maoli/Popoulu, Pisang Raja, Pome, and Mysore, to Foc TR4 under both greenhouse and field conditions. Genotypes from the Silk and Iholena subgroups were highly susceptible to Foc TR4. Pome and Mysore showed resistance and intermediate resistance, respectively. However, Pisang Raja ranged from susceptible to intermediate resistance. One cultivar from the Maia Maoli/Popoulu subgroup was highly susceptible, while the other displayed significant resistance. Most Plantain cultivars exhibited high resistance to Foc TR4, except two French types of cultivar, ‘Uganda Plantain’ and ‘Njombe N°2’, which were susceptible. The susceptibility to Foc TR4 of some of the AAB genotypes evaluated, especially Plantain and other cooking bananas, indicates that growers dependent on these varieties need to be included as part of the prevention and integrated Foc TR4 management strategies, as these genotypes play a crucial role in food security and livelihoods.

Professor Elizabeth Aitken presents her group's work on Fusarium wilt, particularly on banana where host resistance, pathogen diversity and etiology are being explored for the Queensland Chapter of the Australasian Plant Pathology Society (APPS).

Fusarium is one of the most important fungal genera of plant pathogens that affect the cultivation of a wide range of crops. Agricultural losses caused by Fusarium oxysporum f. sp. cubense (Foc) directly affect the income, subsistence, and nourishment of thousands of farmers worldwide. For Viet Nam, predictions on the impact of Foc for the future are dramatic, with an estimated loss in the banana production area of 8% within the next five years and up to 71% within the next 25 years. In the current study, we applied a combined morphological-molecular approach to assess the taxonomic identity and phylogenetic position of the different Foc isolates collected in northern Viet Nam. In addition, we aimed to estimate the proportion of the different Fusarium races infecting bananas in northern Viet Nam. The morphology of the isolates was investigated by growing the collected Fusarium isolates on four distinct nutritious media (PDA, SNA, CLA, and OMA). Molecular phylogenetic relationships were inferred by sequencing partial rpb1, rpb2, and tef1a genes and adding the obtained sequences into a phylogenetic framework. Molecular characterization shows that c. 74% of the Fusarium isolates obtained from infected banana pseudostem tissue belong to F. tardichlamydosporum. Compared to F. tardichlamydosporum, F. odoratissimum accounts for c.10% of the Fusarium wilt in northern Viet Nam, demonstrating that Foc TR4 is not yet a dominant strain in the region. Fusarium cugenangense – considered to cause Race 2 infections among bananas – is only found in c. 10% of the tissue material that was obtained from infected Vietnamese bananas. Additionally, one of the isolates cultured from diseased bananas was phylogenetically not positioned within the F. oxysporum species complex (FOSC), but in contrast, fell within the Fusarium fujikuroi species complex (FFSC). As a result, a possible new pathogen for bananas may have been found. Besides being present on several ABB ‘Tay banana’, F. tardichlamydosporum was also derived from infected tissue of a wild Musa lutea, showing the importance of wild bananas as a possible sink for Foc.

Fusarium oxysporum f.sp. cubense, causal agent of Panama disease, is one of the biggest threats to global banana production, particularly the Cavendish competent tropical race 4 (Foc TR4). It continues to spread globally with detections occurring in regions of the Middle East and new continents such as Africa and South America in the last decade. As the search was on for new management strategies and resistant cultivars to combat the disease, a banana cultivar-screening trial took place in the Northern Territory of Australia, which examined the responses of 24 banana cultivars to the soil borne fungus. These cultivars included material from TBRI, FHIA and selections from Thailand, Indonesia and Australia and evaluated for their resistance to tropical race 4 for two cropping cycles. Several cultivars displayed considerable resistance to Foc TR4, including several FHIA parental lines and hybrids, the Cavendish (AAA) selections GCTCV 215 and GCTCV 247 from TBRI and an Indonesian selection CJ19 showed either very little to no plant death due to the disease.

The socio-economic impact of Fusarium odoratissimum, which is colloquially called tropical race 4 (TR4), is escalating as this fungal pathogen spreads to new banana-growing areas. Hence, the development of simple, reliable and rapid detection technologies is indispensable for implementing quarantine measures. Here, a versatile loop-mediated isothermal amplification (LAMP) assay has been developed that is applicable under field and laboratory conditions. DNA markers unique to TR4 isolates were obtained by diversity arrays technology sequencing (DArTseq), a genotyping by sequencing technology that was conducted on 27 genotypes, comprising 24 previously reported vegetative compatibility groups (VCGs) and three TR4 isolates. The developed LAMP TR4 assay was successfully tested using 22 TR4 isolates and 45 non-target fungal and bacterial isolates, as well as on infected plants under greenhouse and field conditions. The detection limit was 1 pg µL−1 pure TR4 DNA or 102 copies plasmid-localized TR4 unique sequence (SeqA) per reaction, which was not affected by background DNA in complex samples. The LAMP TR4 assay offers a powerful tool for the routine and unambiguous identification of banana plants infected with TR4, contributing to advanced diagnosis in field situations and monitoring of fusarium wilt.

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt or Panama disease on banana, is one of the major constraints in banana production worldwide. Indonesia is the centre of origin for wild and cultivated bananas, which likely co-evolved with Foc. This study explored the widest possible genetic diversity of Foc by sampling across Indonesia at 34 geographically and environmentally different locations in 15 provinces at six islands. This resulted in a comprehensive collection of ∼200 isolates from 40 different local banana varieties. Isolates were identified and assessed using sequence analysis of the translation elongation factor-1alpha (tef1), the RNA polymerase II largest subunit (rpb1), and the RNA polymerase II second largest subunit (rpb2). Phylogenetic analyses of these genes allowed the identification of 180 isolates of Fusarium oxysporum f. sp. cubense (Foc), and 20 isolates of the Fusarium fujikuroi species complex (FFSC), the Fusarium incarnatum-equiseti species complex (FIESC), and the Fusarium sambucinum species complex (FSSC). Further analyses, incorporating a worldwide collection of Foc strains, revealed nine independent genetic lineages for Foc, and one novel clade in the Fusarium oxysporum species complex (FOSC). Selected isolates from each lineage were tested on the banana varieties Gros Michel and Cavendish to characterise their pathogenicity profiles. More than 65 % of the isolates were diagnosed as Tropical Race 4 (Foc-TR4) due to their pathogenicity to Cavendish banana, which supports the hypothesis that Foc-TR4 is of Indonesian origin. Nine independent genetic lineages for Foc are formally described in this study. This biodiversity has not been studied since the initial description of Foc in 1919. This study provides a detailed overview of the complexity of Fusarium wilt on banana and its diversity and distribution across Indonesia. Key words: Morphology, New species, Panama disease, Pathogenicity, Tropical Race 4, 11 New taxa, Taxonomic novelties: New species: Fusarium cugenangense N. Maryani, L. Lombard, Kema & Crous, F. duoseptatum N. Maryani, L. Lombard, Kema & Crous, F. grosmichelii N. Maryani, L. Lombard, Kema & Crous, F. hexaseptatum N. Maryani, L. Lombard, Kema & Crous, F. kalimantanense N. Maryani, L. Lombard, Kema & Crous, F. odoratissimum N. Maryani, L. Lombard, Kema & Crous, F. phialophorum N. Maryani, L. Lombard, Kema & Crous, F. purpurascens N. Maryani, L. Lombard, Kema & Crous, F. sangayamense N. Maryani, L. Lombard, Kema & Crous, F. tardichlamydosporum N. Maryani, L. Lombard, Kema & Crous, F. tardicrescens N. Maryani, L. Lombard, Kema & Crous

Aim Crop wild relatives (CWR) are an essential source of genetic material for the improvement of certain traits in related crop species. Despite their importance, increasing public, scientific and political support, large gaps exist in the amount of genetic material collected and conserved of many CWR. Here, we construct a dataset on the distribution of wild banana species (Musa spp.) and assess their risk and conservation status. We deal with the following questions: (a) What areas are potentially suitable for wild banana species? (b) How much of the wild banana diversity is currently at risk or insufficiently conserved ex and in situ?Location Native distribution area of wild banana species, ranging from the north-eastern states of India to north-eastern Australia.Methods We assessed the potential environmental range of wild species using a species distribution modelling approach with MaxEnt. Extinction risk was evaluated following IUCN criterion B, and the ex and in situ conservation status was assessed using an indicator for biodiversity and sustainable development targets.Results We found that 11 out of 59 assessed species can be considered as vulnerable and nine as endangered. Highest species richness was found along the border of south China and northern Vietnam, in the north-eastern states of India and on the Malayan peninsula. Our distribution modelling approach indicates that the northern Indo-Burmese region has the highest environmental suitability for most wild banana species and that lowland rain forests in general are highly suitable for bananas. Assessment of in and ex situ conservation status indicates that 56 out of 59 assessed species are currently insufficiently conserved ex situ and that 49 are of high priority for further conservation. Additional in situ conservation is of high priority for six species and of medium priority for 40 species.Main conclusions To date, little of the banana CWR are sufficiently conserved both in and ex situ.

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&mdash, including phylogenetics, mating type, vegetative compatibility group (VCG), and pathogenicity testing&mdash, 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 &lsquo, Gros Michel&rsquo, 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 &lsquo, 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.

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.

Bananas are Australia’s number-one selling supermarket product (in volume), with over five million of them eaten daily, all of which are grown in Australia. The 13,000 ha industry has a farm gate value of around $600 m and contributes $1.3 billion to the national economy annually. Over the last 3–4 years the industry has been under considerable financial pressure from poor prices but this has been exacerbated by dealing with major responses to banana Freckle and Fusarium Wilt Tropical Race 4 (TR4). Banana Freckle has successfully been eradicated from Australia but the threat from TR4 is ongoing. The Banana industry considers that R&D is critically important for successfully dealing with major disease threats and reducing their impact. The spread of TR4 has been slowed through a major control and containment program which has been underpinned by high quality research but this research needs to continue, to allow time to find long-term solutions that will safeguard the industry’s ongoing viability. The role science plays is multifaceted. The challenge for horticultural industries is to include both basic and applied research that is relevant to the needs of industry, fits within the constraints of legislation and the supply chain, meets the needs of consumers and will lead to outcomes that are implemented on farms by growers. Examples of how the banana industry is meeting these challenges will be discussed in this presentation.

Panama wilt, caused by Fusarium oxysporum f. sp. cubense (Foc) is considered one of the most devastating banana diseases in recorded history. The disease threatens the banana industry due to Tropical Race 4 (TR4) infecting the Cavendish cultivar. Forty-two of the 45 representative isolates from Luzon were pathogenic, based on leaf symptom index and vascular discoloration rating. Accurate, fast and reliable identification are pre-requisites for effective management considering there are yet no proven effective chemicals to control the disease, thus the confirmation by a PCR-based diagnostic tool is essential. Using race-specific primers, FocTr4-F/FocTr4-R and Foc-1/Foc-2, the absence of TR4 in Luzon has been confirmed, however, the occurrence of Race 4 has been reported, which should also be taken in consideration as the latter can also cause severe damage under favorable conditions. Furthermore, to examine genetic diversity of Foc in bananas, 55 of the 164 isolates collected from Regions I, II, III, IV and Cordillera Administrative Region (CAR) were analyzed by fingerprinting techniques using M13, ERIC and REP primers. Twenty-two reference isolates from Mindanao were also analyzed using the same primers. Foc isolates were differentiated into two clades at 25% similarity level, classifying all Mindanao isolates to clade A. Consistently high genetic variation was obtained from Luzon isolates using M13, an arbitrarily primed fingerprinting technique and repetitive elements, REP and ERIC-PCR, while low genetic variation was obtained from Mindanao isolates. ERIC-PCR was the most informative and predictive fingerprinting method as the TR4 isolates from Mindanao were grouped together. No grouping of Foc isolates was observed with respect to geographical origin, except isolates from Mindanao. In addition, grouping of Foc4 is also regardless of host variety in all analyses conducted. Overall, high genetic variability was recorded in Foc Philippine population for the three primers used, which might render host resistance vulnerable.

Panama wilt, caused by Fusarium oxysporum f. sp. cubense (Foc) is considered one of the most devastating banana diseases in recorded history. The disease threatens the banana industry due to Tropical Race 4 (TR4) infecting the Cavendish cultivar. Forty-two of the 45 representative isolates from Luzon were pathogenic, based on leaf symptom index and vascular discoloration rating. Accurate, fast and reliable identification are pre-requisites for effective management considering there are yet no proven effective chemicals to control the disease, thus the confirmation by a PCR-based diagnostic tool is essential. Using race-specific primers, FocTr4-F/FocTr4-R and Foc-1/Foc-2, the absence of TR4 in Luzon has been confirmed, however, the occurrence of Race 4 has been reported, which should also be taken in consideration as the latter can also cause severe damage under favorable conditions. Furthermore, to examine genetic diversity of Foc in bananas, 55 of the 164 isolates collected from Regions I, II, III, IV and Cordillera Administrative Region (CAR) were analyzed by fingerprinting techniques using M13, ERIC and REP primers. Twenty-two reference isolates from Mindanao were also analyzed using the same primers. Foc isolates were differentiated into two clades at 25% similarity level, classifying all Mindanao isolates to clade A. Consistently high genetic variation was obtained from Luzon isolates using M13, an arbitrarily primed fingerprinting technique and repetitive elements, REP and ERIC-PCR, while low genetic variation was obtained from Mindanao isolates. ERIC-PCR was the most informative and predictive fingerprinting method as the TR4 isolates from Mindanao were grouped together. No grouping of Foc isolates was observed with respect to geographical origin, except isolates from Mindanao. In addition, grouping of Foc4 is also regardless of host variety in all analyses conducted. Overall, high genetic variability was recorded in Foc Philippine population for the three primers used, which might render host resistance vulnerable.

Fusarium oxysporum f.sp. cubense, causal agent of Panama disease, is one of the biggest threats to global banana production, particularly the Cavendish competent tropical race 4 (Foc TR4). It continues to spread globally with detections occurring in regions of the Middle East and new continents such as Africa and South America in the last decade. As the search was on for new management strategies and resistant cultivars to combat the disease, a banana cultivar-screening trial took place in the Northern Territory of Australia, which examined the responses of 24 banana cultivars to the soil borne fungus. These cultivars included material from TBRI, FHIA and selections from Thailand, Indonesia and Australia and evaluated for their resistance to tropical race 4 for two cropping cycles. Several cultivars displayed considerable resistance to Foc TR4, including several FHIA parental lines and hybrids, the Cavendish (AAA) selections GCTCV 215 and GCTCV 247 from TBRI and an Indonesian selection CJ19 showed either very little to no plant death due to the disease.

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.

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.

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt or Panama disease on banana, is one of the major constraints in banana production worldwide. Indonesia is the centre of origin for wild and cultivated bananas, which likely co-evolved with Foc. This study explored the widest possible genetic diversity of Foc by sampling across Indonesia at 34 geographically and environmentally different locations in 15 provinces at six islands. This resulted in a comprehensive collection of ∼200 isolates from 40 different local banana varieties. Isolates were identified and assessed using sequence analysis of the translation elongation factor-1alpha (tef1), the RNA polymerase II largest subunit (rpb1), and the RNA polymerase II second largest subunit (rpb2). Phylogenetic analyses of these genes allowed the identification of 180 isolates of Fusarium oxysporum f. sp. cubense (Foc), and 20 isolates of the Fusarium fujikuroi species complex (FFSC), the Fusarium incarnatum-equiseti species complex (FIESC), and the Fusarium sambucinum species complex (FSSC). Further analyses, incorporating a worldwide collection of Foc strains, revealed nine independent genetic lineages for Foc, and one novel clade in the Fusarium oxysporum species complex (FOSC). Selected isolates from each lineage were tested on the banana varieties Gros Michel and Cavendish to characterise their pathogenicity profiles. More than 65 % of the isolates were diagnosed as Tropical Race 4 (Foc-TR4) due to their pathogenicity to Cavendish banana, which supports the hypothesis that Foc-TR4 is of Indonesian origin. Nine independent genetic lineages for Foc are formally described in this study. This biodiversity has not been studied since the initial description of Foc in 1919. This study provides a detailed overview of the complexity of Fusarium wilt on banana and its diversity and distribution across Indonesia.

Following a request from the European Commission, the Panel on Plant Health was asked to deliver a scientific opinion on 30 pest risk assessments made by France on organisms which are considered by France as harmful in four French overseas departments, i.e. French Guiana, Guadeloupe, Martinique and Réunion. In particular, the Panel was asked whether these organisms can be considered as harmful organisms for the endangered area of the above departments, in the meaning of the definition mentioned in Article 2.1. (e) of Directive 2000/29/EC and thus potentially eligible for addition to the list of harmful organisms in Directive 2000/29/EC.This document presents the opinion of the Panel on Plant Health on the full[2] pest risk assessment conducted by France on Fusarium oxysporum Schlecht. f. sp. cubense (E.F. Smith) W.C. Snyder & H.S. Hansen with French Guiana, Guadeloupe, Martinique and Réunion considered as endangered area.F. oxysporum f. sp. cubense is responsible for Fusarium wilt (also known as Panama disease), which is one of the most destructive diseases of banana world-wide. Control of the disease is restricted to husbandry measures and cultivar resistance.F. oxysporum f. sp. cubense has been divided into races based on its pathogenicity on reference host cultivars. Among the most significant races of F. oxysporum f. sp. cubense pathogenic to edible banana, race 1 affects mainly the cultivar ‘Gros Michel’, race 2 affects bananas of the Bluggoe sub-group, race 4 affects all cultivars in the Cavendish sub-group grown in subtropical climates plus cultivars susceptible to races 1 and 2. F. oxysporum f. sp. cubense race 4 is commonly known as ‘subtropical race 4’. F. oxysporum f. sp. cubense ‘tropical race 4’ (hereinafter T4) is a new variant, which was recognised in the early 1990s and is responsible for serious losses of cultivars in the Cavendish subgroup in some areas of tropical South East Asia.Both races 1 and 2 are reported to be endemic in the PRA area[3], while races 4 and T4 are not known to be present. Race 4 has been under quarantine regulation since 1992 for French Guiana, Guadeloupe, Martinique and Réunion.The Panel examined in detail the risk assessment provided, and considered the accuracy and quality of the information provided and methods applied for pest risk assessment purposes. The review was based on the principles of the International Standard on Phytosanitary Measures ISPM No. 11[4]: Pest risk analysis for quarantine pests, including analysis of environmental risks and living modified organisms (2004) by the International Plant Protection Convention (FAO, 2007b).Following a detailed analysis of the material provided in the pest risk assessment and after additional study of published information on the pathogen and the disease it causes, the Panel observes that:*The document only cites one reference, which is a review article. As a consequence, statements in the pest risk assessment are not individually referenced or supported by verifiable scientific data.*The risk assessor identifies the pest as F. oxysporum f.sp. cubense, which implies that all races are under consideration. However most of the document focuses only on races4 and T4. This confuses the identity of the subject organism of the pest risk assessment.*F. oxysporum f.sp. cubense, races 1 and 2 are reported to be present in most departments defined as the PRA area (i.e. French Guiana, Guadeloupe and Martinique) and are not reported to be under official control in the document. Races 4 and T4 are not known to be present in the PRA area.The Panel concludes that:*Only the races 4 and T4 fulfil the criteria for a quarantine pest[5], as they are not known to occur in the PRA area and have the potential for establishment and spread and for negative economic consequences in the PRA area.*In agreement with the document, the probability of entry of F. oxysporum f.sp. cubense races 4 and T4 into the PRA area on conventional planting material, from countries where the disease is present, would be high if present import restrictions were rescinded.*In agreement with the document, the probability of establishment for F. oxysporum f.sp. cubense race 4, which attacks Cavendish cultivars in the subtropics, would be low, except for those parts of the PRA area with subtropical climate, where the probability for establishment of race 4 would be high.*The probability of establishment for F. oxysporum f.sp. cubense race T4, which is able to attack Cavendish cultivars in the tropics, would be high (rather than “moderate”), as most of the endangered area has a tropical climate and no commercial Cavendish cultivars resistant to race T4 have yet been developed or tested in the French overseas departments.*In agreement with the document, the potential economic impacts of races 4 and T4 would be ’high’. Plantations of dessert bananas for export and smallholdings and family gardens producing fruit for the local market or for home-consumption would suffer severe yield losses. Concerning social impacts, for Guadeloupe and Martinique there could be a reduction in employment in commercial plantations, whilst a potential disruption of subsistence production and consumption patterns could occur among smallholders and family gardens.The Panel, based on the information provided in the document and on additional literature consulted, concludes that F. oxysporum f.sp. cubense races 4 and T4 are appropriate for evaluation of pest risk management options for the endangered areas of French Guiana, Guadeloupe, Martinique and Réunion, and thus potentially eligible for addition to the list of harmful organisms in Directive 2000/29/EC.