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Bacterial Wilt of Banana Diagnostics Manual

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Bacterial Wilt of Banana Diagnostics Manual Bacterial Wilt of Banana Diagnostics Manual 1 Bacterial Wilt Diagnostics Manual 2006 2 Table of Contents 1 Introduction.........................................................................................................5 1.1. The Banana Industry in Australia ................................................................. 5 1.2. History of the Diseases ................................................................................ 5 1.3. Global Distribution of the Diseases .............................................................. 8 1.4. The disease in Australia............................................................................... 9 2 Disease Symptoms ...........................................................................................10 2.1 Moko disease............................................................................................. 10 2.2 Bugtok........................................................................................................ 13 2.3 Blood disease ............................................................................................ 14 The Pathogens .......................................................................................................15 2.4 The Causal Agent ...................................................................................... 15 2.5 Taxonomy .................................................................................................. 15 2.5.1 Diversity of the R. solanacearum species complex............................. 15 3.2.2 The R. solanacearum species complex .............................................. 16 3.2.3 The Phylotyping scheme: a new scheme for classifying R. solanacearum.................................................................................................... 17 2.5.2 Comparison of the phylotyping classification schemes....................... 19 3.3 Taxonomy and Diversity of R. solanacearum race 2 strains ...................... 21 3.3.2 Moko and Bugtok diseases in the Philippines............................................. 23 3.4 Taxonomy and Diversity of BDB strains..................................................... 24 3.5 Hosts.......................................................................................................... 26 4 Disease Development and Epidemiology .......................................................28 4.2 Disease Cycle............................................................................................ 28 4.3 Survival of the Inoculum............................................................................. 28 5 Disease Control.................................................................................................30 5.1 Cultural Control............................................................................................... 30 5.2 Chemical Control ........................................................................................... 30 6 Molecular Diagnosis of Diseases ....................................................................31 6.1 Introduction ................................................................................................ 31 6.2 Preparation of Samples to be tested by PCR............................................. 31 6.2.1 Materials ............................................................................................. 31 6.2.2 Sampling Protocols............................................................................. 32 6.3 R. solanacearum race 2/biovar 1 PCR protocols ....................................... 34 6.3.1 Solutions required............................................................................... 34 6.3.2 Setting up individual PCR reactions.................................................... 34 6.3.3 Multiplex PCR reaction ....................................................................... 37 6.3.4 Gel electrophoresis............................................................................. 38 6.4 Precautions During PCR Assays ............................................................... 39 6.5 Preferred Molecular Protocol ..................................................................... 40 6.5.1 Preparation of template for PCR......................................................... 40 6.5.2 Setting up the multiplex PCR reaction ................................................ 41 6.5.3 PCR Protocol ...................................................................................... 41 6.5.4 Gel electrophoresis............................................................................. 42 3 7 BDB PCR ...........................................................................................................43 8 Phylotype multiplex PCR .................................................................................45 9 Troubleshooting................................................................................................48 9.1 Troubleshooting Guide 1 - Gel Electrophoresis .............................................. 48 9.2 Troubleshooting Guide 2 – PCR Diagnostic Results ...................................... 50 9.3 Limitations of the Technology .................................................................... 52 10 Sources of Reference Material.........................................................................54 10.1 DNA/Bacterial Cultures................................................................................. 54 11 Media and Stock Solutions ..............................................................................55 11.1 Media............................................................................................................ 55 11.1.1 Sucrose Peptone Agar (SPA)................................................................. 55 11.1.2 Modified – SMSA ................................................................................ 55 11.2 Stock Solutions .......................................................................................... 56 11.2.1 6X Loading Buffer ............................................................................... 56 11.2.2 Tris-Acetate-EDTA buffer – 50X stock ................................................... 56 12 Further Reading ................................................................................................57 13 References ........................................................................................................58 4 1 Introduction 1.1. The Banana Industry in Australia In 2003 the farm gate value of the Australian banana industry was $295 million dollars. This resulted from more than 1900 growers producing in excess of 300,000 tonnes of bananas from 15,500 Ha. More than 80% of this crop is produced in the wet tropics of Far North Queensland. The remaining 20% are grown predominantly in the subtropical regions of the eastern coast from Nambucca Heads, in New South Wales to Nambour in Queensland. There are also small producing regions in Darwin in the Northern Territory and also Kununurra and Carnarvon in Western Australia. Approximately 95% of bananas grown in Australia are of the Cavendish subgroup with the remaining 5% comprising mainly the Ladyfinger cultivar but also some Goldfinger, Ducasse, Sucrier, FHIA 18, Red Dacca and Plantains. In Australia sales of banana are mostly that of the fresh fruit product. The market is mature and as such, notwithstanding changes to current trends, should follow population growth. Currently the only other products from banana fruit and fibre are the small-scale production of banana puree, dried banana, banana wine and handbags. Although at this time there is little value added product emanating from the banana industry the development of a paper production facility at Mareeba, utilising the pseudostems of banana plants to provide the long fibres required for the paper production, has generated much interest by both the local council and also growers interested in supplying the pseudostems. 1.2. History of the Diseases Although the three diseases Moko, Bugtok and Blood disease are closely related the distribution of each is quite distinct (Figure 1). All three however are limited to the tropics. Moko which has the widest distribution of the three has not been verified as being present in any regions outside of the area between latitudes 18º N and 18º S. Moko disease was first reported in Trinidad in the 1890s when it caused severe losses of Moko cooking bananas (ABB genotype, syn Bluggoe). The causal agent of the disease was described in 1911 by Rorer. Because this initial outbreak of the 5 disease was so severe the cultivation of Moko cooking bananas was completely abandoned. Due to this, outbreaks of Moko disease were no longer as devastating until the disease reached the large plantations of dessert bananas planted in the 1940s and 1950s when again severe losses were experienced. Much research carried out after this time has lead to good control measures being adopted within these commercial plantings resulting in low disease incidence. Moko disease does still affect the smallholder farmers however and so has subsequently spread throughout many parts of Latin America and is also present in the Caribbean. It is officially recorded in Belize, Brazil, Colombia, Costa Rica, Ecuador, El Salvadore, Grenada, Guatemala, Guyana, Honduras, Mexico, Nicaragua, Panama, Peru, Surinam, Trinidad and Venezuela (Stover 1972; Phelps 1987). In 2004 the disease reached the parish of St James in Jamaica. Restrictions have been placed
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