Segmental duplications drive the evolution of accessory regions in a major crop pathogen.

Many pathogens evolved compartmentalized genomes with conserved core and variable accessory regions (ARs) that carry effector genes mediating virulence. The fungal plant pathogen Fusarium oxysporum has such ARs, often spanning entire chromosomes. The presence of specific ARs influences the host range, and horizontal transfer of ARs can modify the pathogenicity of the receiving strain. However, how these ARs evolve in strains that infect the same host remains largely unknown. We defined the pan-genome of 69 diverse F. oxysporum strains that cause Fusarium wilt of banana, a significant constraint to global banana production, and analyzed the diversity and evolution of the ARs. Accessory regions in F. oxysporum strains infecting the same banana cultivar are highly diverse, and we could not identify any shared genomic regions and in planta-induced effectors. We demonstrate that segmental duplications drive the evolution of ARs. Furthermore, we show that recent segmental duplications specifically in accessory chromosomes cause the expansion of ARs in F. oxysporum. Taken together, we conclude that extensive recent duplications drive the evolution of ARs in F. oxysporum, which contribute to the evolution of virulence.

The Vulnerability of Cuban Banana Production to Fusarium Wilt Caused by Tropical Race 4.

Bananas are major agricultural commodities in Cuba. One of the main constraints of banana production worldwide is Fusarium wilt of banana. Recent outbreaks in Colombia, Perú, and Venezuela have raised widespread concern in Latin America due to the potential devastating impact on the sustainability of banana production, food security, and livelihoods of millions of people in the region. Here, we phenotyped 18 important Cuban banana and plantain varieties with two Fusarium strains-Tropical Race 4 (TR4) and Race 1-under greenhouse conditions. These varieties represent 72.8% of the national banana acreage in Cuba and are also widely distributed in Latin America and the Caribbean region. A broad range of disease responses from resistant to very susceptible was observed against Race 1. On the contrary, not a single banana variety was resistant to TR4. These results underscore that TR4 potentially threatens nearly 56% of the contemporary Cuban banana production area, which is planted with susceptible and very susceptible varieties, and call for a preemptive evaluation of new varieties obtained in the national breeding program and the strengthening of quarantine measures to prevent the introduction of TR4 into the country.

Induced resistance to Fusarium wilt of banana caused by Tropical Race 4 in Cavendish cv Grand Naine bananas after challenging with avirulent Fusarium spp.

In the last century, Fusarium wilt of banana (FWB) destroyed the banana cultivar Gros Michel. The Cavendish cultivars saved the global banana industry, and currently they dominate global production (~50%) and the export trade (~95%). However, a new strain called Tropical Race 4 (TR4) surfaced in the late 1960's, spread globally and greatly damages Cavendish plantations as well as manifold local varieties that are primarily grown by small holders. Presently, there is no commercially available replacement for Cavendish and hence control strategies must be developed and implemented to manage FWB. Here, we studied whether it is possible to induce resistance to TR4 by pre-inoculations with different Fusarium spp. Only pre-treatments with an avirulent Race 1 strain significantly reduced disease development of TR4 in a Cavendish genotype and this effect was stable at various nutritional and pH conditions. We then used transcriptome analysis to study the molecular basis of this response. Several genes involved in plant defence responses were up-regulated during the initial stages of individual infections with TR4 and Race 1, as well as in combined treatments. In addition, a number of genes in the ethylene and jasmonate response pathways as well as several gibberellin synthesis associated genes were induced. We observed upregulation of RGA2 like genes in all treatments. Hence, RGA2 could be a key factor involved in both R1 and TR4 resistance. The data support the hypothesis that activating resistance to Race 1 in Cavendish bananas affects TR4 development and provide a first insight of gene expression during the interaction between various Fusarium spp. and banana.

An Improved Phenotyping Protocol for Panama Disease in Banana.

Fusarium oxysporum (Fo) belongs to a group of soil-borne hyphomycetes that are taxonomically collated in the Fusarium oxysporum Species Complex (FOSC). Hitherto, those infecting bananas were placed in the forma specialis cubense (Foc). Recently, however, these genetically different Foc lineages were recognized as new Fusarium spp. placed in the Fusarium of Banana Complex (FOBC). A member of this complex F. odoratissimum II-5 that uniquely comprises the so-called Tropical Race 4 (TR4), is a major problem sweeping through production zones of Cavendish banana in several regions of the world. Because of this, there is an urgent need for a phenotyping method that allows the screening for resistance to TR4 of large numbers of banana genotypes. Most Fusarium species produce three types of spores: macroconidia, microconidia and the persistent chlamydospores that can contaminate soils for many years. Inoculum production has been an important bottleneck for efficient phenotyping due to the low or variable number of conidia and the elaborate laboratory procedures requiring specific infrastructure. Here, we report a rapid, simple and high-yielding spore production method for nine F. oxysporum formae speciales as well as the biocontrol species Fo47 and Fo618-12. For Fusarium spp. causing Fusarium wilt or Panama disease of banana, we used the protocol for four species comprising the recognized physiological races, including Tropical Race 4 (TR4). We subsequently tested the produced inoculum in comparative inoculation trials on banana plants to evaluate their efficiency. All assays resulted in typical symptoms within 10 weeks; significant differences in final disease ratings were observed, depending on inoculum concentration. Pouring inoculum directly onto banana plants showed the most consistent and reproducible results, as expressed in external wilting, internal discoloration and determined by real-time PCR assays on entire rhizomes. Moreover, this method allows the inoculation of 250 plants per hour by one individual thereby facilitating the phenotyping of large mutant and breeding populations.

New Geographical Insights of the Latest Expansion of Fusarium oxysporum f.sp. cubense Tropical Race 4 Into the Greater Mekong Subregion.

Banana is the most popular and most exported fruit and also a major food crop for millions of people around the world. Despite its importance and the presence of serious disease threats, research into this crop is limited. One of those is Panama disease or Fusarium wilt. In the previous century Fusarium wilt wiped out the "Gros Michel" based banana industry in Central America. The epidemic was eventually quenched by planting "Cavendish" bananas. However, 50 years ago the disease recurred, but now on "Cavendish" bananas. Since then the disease has spread across South-East Asia, to the Middle-East and the Indian subcontinent and leaped into Africa. Here, we report the presence of Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4) in "Cavendish" plantations in Laos, Myanmar, and Vietnam. A combination of classical morphology, DNA sequencing, and phenotyping assays revealed a very close relationship between the Foc TR4 strains in the entire Greater Mekong Subregion (GMS), which is increasingly prone to intensive banana production. Analyses of single-nucleotide polymorphisms enabled us to initiate a phylogeography of Foc TR4 across three geographical areas-GMS, Indian subcontinent, and the Middle East revealing three distinct Foc TR4 sub-lineages. Collectively, our data place these new incursions in a broader agroecological context and underscore the need for awareness campaigns and the implementation of validated quarantine measures to prevent further international dissemination of Foc TR4.

Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4.

Banana (Musa spp.) is a staple food for more than 400 million people. Over 40% of world production and virtually all the export trade is based on Cavendish banana. However, Cavendish banana is under threat from a virulent fungus, Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) for which no acceptable resistant replacement has been identified. Here we report the identification of transgenic Cavendish with resistance to TR4. In our 3-year field trial, two lines of transgenic Cavendish, one transformed with RGA2, a gene isolated from a TR4-resistant diploid banana, and the other with a nematode-derived gene, Ced9, remain disease free. Transgene expression in the RGA2 lines is strongly correlated with resistance. Endogenous RGA2 homologs are also present in Cavendish but are expressed tenfold lower than that in our most resistant transgenic line. The expression of these homologs can potentially be elevated through gene editing, to provide non-transgenic resistance.