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Of Banana Caused by Fusarium Oxysporum F Technical Manual Prevention and diagnostic of Fusarium Wilt (Panama disease) of banana caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4) Luis Pérez-Vicente PhD. Senior Plant Pathologist, INISAV, Ministry of Agriculture, Cuba. Expert Consultant on Fusarium wilt disease of banana Miguel A. Dita, PhD. Research Scientist on Plant Pathology, Brazilian Research Agricultural Corporation – EMBRAPA, Brazil. Expert Consultant on Fusarium wilt disease of banana Einar Martínez- de la Parte, MSc Plant Pathologist, INISAV, Ministry of Agriculture, Cuba. Expert Consultant on Fusarium wilt disease of banana Prepared for the Regional Workshop on the Diagnosis of Fusarium Wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense Tropical Race 4: Mitigating the Threat and Preventing its Spread in the Caribbean FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS May 2014 1 Technical Manual Prevention and diagnostic of Fusarium Wilt (Panama disease) of banana caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4) Luis Pérez-Vicente PhD. Senior Plant Pathologist, INISAV, Ministry of Agriculture, Cuba. Expert Consultant on Fusarium wilt disease of banana Miguel A. Dita, PhD. Research Scientist on Plant Pathology, Brazilian Research Agricultural Corporation – EMBRAPA, Brazil. Expert Consultant on Fusarium wilt disease of banana Einar Martínez- de la Parte, MSc Plant Pathologist, INISAV, Ministry of Agriculture, Cuba. Expert Consultant on Fusarium wilt disease of banana Prepared for the Regional Workshop on the Diagnosis of Fusarium Wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense Tropical Race 4: Mitigating the Threat and Preventing its Spread in the Caribbean FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS May 2014 2 INDEX Introduction:……………………………………………………………………………………………………………………………………4 Fusarium wilt of banana or panama disease by Fusarium oxysporum f. Sp. cubense: a review on history, symptoms, biology, epidemiology and management………………....……………………….................6 Introduction………………………….…………………………………………………………………………………….…………………….…………….6 Fusarium wilt symptoms….………………………………………………………………………………….……………………………..………..7 Causal agent: notes on taxonomy and nomenclature ................................................................................... 9 Morphology and anatomy .………………………………………..……………………………………………………………...11 Pathogen variability………………………………………………………………………………………………………….…..……12 Biology and ecology.......…………………………………………………………………………………………………………....13 Hosts …………………………………………………………………………………………………………………………………………14 Geographic distribution…………………………………………………………………………………………….……............16 Fusarium wilt impact and damages……………………………………………………………………………….………… 17 Phytosanitary risks ………………………………………………………………………………………………………..…..…..…18 Risk management …………………………………………………………………………………………………..…………………19 Erradication of an outbreak of Foc TR4 …………………………………………………………………..……….…….….20 Disease management……………………………………………………………………………………………..………….….....22 References …………………………………………………………………………….…………………………………..…….…......23 PROTOCOLS……………………………………………………………………………………………………………………….….…....32 Protocol for sampling, transport and storage of samples……………………………………………..………..….33 Protocol for the isolation of Fusarium oxysporum f. sp. cubense……………………….…..…..………….....36 Protocol for determination of vegetative compatibility groups (VCG’s)………………..…………………...41 Protocol for Fusarium oxysporum cultures storage………………………………………………..………….……...54 Inoculation of Fusarium oxysporum f. sp. cubense in banana………………………………..…………….…….57 Protocols for DNA extraction…………………………………………………………………………………..………….……..61 Protocols for molecular identification of Fusarium oxysporum f. sp. cubense………..……….………….66 3 INTRODUCTION Global banana production is seriously threatened by the re-emergence of a Fusarium Wilt. The disease, caused by the soil-borne fungi Fusarium oxysporum f. sp. cubense (Foc) and also known as “Panama disease”, wiped out the Gros Michel banana industry in Central America and the Caribbean, in the mid-twentieth century. The effects of Foc Race 1 were overcome by a shift to resistant Cavendish cultivars, which are currently the source of 99% of banana exports. Unfortunately, a new strain of Foc called Tropical race 4 (TR4) has overcome Foc resistance in Cavendish clones. Perhaps even more seriously, other banana cultivars such as plantains, cooking bananas and a diverse range of dessert banana varieties (not susceptible to races 1 and 2) are also susceptible to TR4. These local varieties include are mostly grown by smallholder farmers for local consumption and income generation. More than 80% of global banana and plantain production is thought to be based on TR4 susceptible germplasm. This strain of Foc has caused epidemics in Cavendish in the tropics different from those less-severe infections previously reported in the sub-tropics. This brings back memories of the devastating damage caused by R1 that led to losses estimated at more than a billion dollars during that era. The devastating impact of Fusarium wilt on Cavendish plantations in Asia was first observed in Taiwan in the late 1960s, which eventually caused a significant reduction of production to just 10% of former levels, and had caused significant increases in production costs rendering its exports much less competitive. In the early 1990s, thousands of hectares of Indonesian and Malaysian Cavendish commercial plantations failed to establish due to severe epidemics of TR4, causing hundreds of millions of dollars in production losses, including from those cultivars grown by smallholder growers. The occurrence of TR4 epidemics in Cavendish farms in China (2004) and the Philippines (2008) and more recently in Mozambique (2013) has renewed serious concerns with regard to its destructive potential in the tropics where most bananas for export and local consumption are produced. It now threatens the 400 million-dollar banana export industry of the Philippines, currently the second largest supplier of the global market after Ecuador. It is also spreading and causing damage to the predominantly Cavendish-based banana production in China, which is presently the third largest banana producer of the world after India and Brazil. Preliminary risk analysis indicated that the spread of TR4 to Africa and the Americas was only a question of time. The recent wide TR4 outbreaks reported in Oman, Jordan, Pakistan (under evaluation), and Mozambique during 2013 has proven its threat as a trans-boundary disease of special importance to other major banana producing countries in the world. This trans-boundary phenomenon threatens not only the multimillion-dollar banana export industry, but also millions of people in rural communities, who depend on bananas for their food security and livelihoods. Planting material, water, soil particles, tools footwear and machineries can efficiently disseminate the pathogen. The fungus can survive in soil for more than 20 years, has a long latent period (it might be detected long time after the introduction) and there is no symptomatic differences among races. Besides this, cultural practices and socioeconomic factors that contribute to Race 1 epidemic still present and would contribute to a TR4 epidemic if the pathogen reaches Latin America and the Caribbean (LAC). Early detection of symptoms in the field and fast laboratory diagnostic is an essential step to either eradication or containment an eventual outbreak. 4 Aware on the potential threat of TR4 to LAC, MUSALAC (The Latin American Network for Research and Development of Musa) and Bioversity International embarked on many awareness campaigns since 2004 about TR4’s threat. Raising awareness was stated as a key step to prevent the introduction of this pathogen in LAC, a highly dependent region on banana and plantains. In 2009, an expert meeting was carried out at the headquarters of the Regional Organisation for Plant and Animal Health (OIRSA) in El Salvador. As a consequence of this meeting, specific legislations on phytosanitary and quarantine were adopted by different countries in the region. In addition, training courses and capacity building workshops on the disease have been developed. These events included relevant aspects on Fusarium wilt such as symptoms recognition, sampling procedures, pathogen diagnosis as well as available options for eradication and disease management. The first training course was carried out in Costa Rica in 2009 and subsequently Cuba (2010), México (2011), Colombia (2011, 2012), Ecuador (2011, 2013), Peru (2011), Nicaragua (2011), Puerto Rico (2013) and Dominican Republic (2013) were capacitated. In all the cases, the National Plant Protection Organizations (NPPOs) and different government agencies were involved. As a complement to the aforementioned initiatives, a Contingency Plan for an eventual TR4 outbreak was recently developed by OIRSA, with the participation of researchers of Bioversity International, INISAV and an OIRSA expert. This document offers guidelines to NPPOs on how to deal with a putative incursion of TR4 in the region. The document has open access, can be consulted on OIRSA’s web site and will be provided to the participants of this Workshop. Fusarium wilt is currently considered (with some exceptions) a minor disease in LAC. Many of the persons that dealt with the first epidemic of race 1 in the past are no longer alive or are long retired. Therefore, there is a lack of capacity to deal with this disease at different levels and even with the
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