Barley Stripe Rust National Diagnostic Protocol Merrin Spackman Department of Primary Industries Primary Industries Research Victoria, Horsham. July 2005 ACKNOWLEDGMENTS Plant Health Australia funded the project to develop this manual as part of their National Diagnostic Protocols Initiative. Special acknowledgment is given to Dr Manilal William, CIMMYT Mexico, for diagnostic images and Dr Colin Wellings, University of Sydney, Plant Breeding Institute, Cobbitty, for diagnostic images and intellectual input. DISCLAIMER The scientific and technical content of this document is current to the date published and all efforts were made to obtain relevant and published information on the pest. New information will be included as it becomes available, or when the document is reviewed. The material contained in this publication is produced for general information only. It is not intended as professional advice on any particular matter. No person should act or fail to act on the basis of any material contained in this publication without first obtaining specific, independent professional advice. Plant Health Australia and all persons acting for Plant Health Australia in preparing this publication, expressly disclaim all and any liability to any persons in respect of anything done by any such person in reliance, whether in whole or in part, on this publication. The views expressed in this publication are not necessarily those of Plant Health Australia. Contents 1.0 Introduction 1.1 Description 1.2 Spread 1.3 Strains 1.4 Hosts 1.5 Losses 1.6 Control 2.0 National Diagnostic Protocol Procedure 2.1 Purpose and scope of diagnostic protocol 2.2 Responsibility 2.3 Procedure 2.4 Documentation 2.5 Records 3.0 Pest Risk Analysis 3.1 Background 3.2 Species name 3.3 Synonyms 3.4 Common names 3.5 Host Range 3.6 Distribution 3.6.1 Current distribution 3.6.2 Australian status 3.6.3 Potential distribution in Australia 3.7 Plant parts affected 3.7.1 Vegetative 3.7.2 Seedborne 3.8 Disease features 3.9 Biology 3.9.1 Identification 3.9.2 Symptoms 3.9.3 Disease cycle 2 3.9.4 Dispersal 3.10 Assessment of likelihood 3.10.1 Entry potential 3.10.2 Establishment potential 3.10.3 Spread potential 3.11 Overall entry, establishment and spread potential 3.12 Assessment of consequences 3.12.1 Economic impact 3.12.2 Environmental impact 3.12.3 Social impact 3.13 Combination of likelihood and consequences to assess risks 3.14 Surveillance 3.15 Diagnostics 3.16 Training 4.0 Diagnostic protocol 4.1 The diagnostic test/s and diagnostic sequence 4.2 The initial samples 4.2.1 Sample handling and subsampling 4.2.2 Sample storage 4.2.3 Visual symptoms 4.2.4 Documentation 4.3 Further samples 4.3.1 Sample collection, transport and storage 4.3.2 Sample locations 4.4 Confirmation of diagnosis 5.0 Identification of pathogen (primary diagnostic test) 5.1 PCR test 5.1.2 DNA Extraction 5.1.2.1 General items required 5.1.2.2 Method 5.1.3 Detection 5.1.3.1 Items required 5.1.3.2 Primers 3 5.1.3.3 PCR controls 5.1.3.4 PCR reagents 5.1.3.5 PCR program 5.1.3.6 Electrophoresis 5.1.3.7 Results 5.1.3.8 Recipes 5.1.3.9 Ordering information 6.0 Confirmation of diagnosis 6.1 Confirmatory (secondary diagnostic) test 6.1.1 Introduction 6.1.2 General items required 6.1.3 Specific items 6.1.4 Method 7.0 Images 8.0 References and websites 8.1 References 8.2 Websites 9.0 Appendices Appendix 1. Preliminary Information Data Sheet (Plantplan, 2004) Appendix 2. Personnel Hygiene Appendix 3. Machinery Hygiene 4 List of Figures Figure 1. Life Cycle.. Figure 2. Flow diagram of the protocols for the analysis of a suspect plant sample. Figure 3. Flow chart of protocols for the diagnosis of suspect barley stripe rust-infected plants. Figure 4. The potential distribution of barley stripe rust in Australia. Figure 5. Amplification products using SSR primers RJ18. Figure 6. Amplification products using SSR primers RJ24. Figure 7. Pressurised spray gun for distributing spores. Figure 8. Humidity chambers with mister in cool room. Figure 9. The stripes of stripe rust. Figure 10. Dr C. Wellings assessing a field trial of barley stripe rust at CIMMYT. Figure 11. Infection of P.s. hordei on highly susceptible barley. Figure 12. Striping infection type typical of P.s. hordei infection. List of Tables Table 1. Host range of P. striiformis f.sp. hordei. Table 2. World distribution of P. striiformis f.sp. hordei. Table 3. Differences in colour, infection position and pattern between the cereal rust diseases. Table 4. Barley varieties used as differential testers for diagnosis of barley stripe rust. Table 5. Scale of infection for rust symptoms. 5 1.0 Introduction Rust diseases have caused sporadic crop losses in most barley producing regions of the world. Stripe rust, caused by Puccinia striiformis exists in several biological forms (formae speciales) that vary in host range between and within genera and species of the Gramineae family (Stubbs, 1985). The pathogen already occurs in Australia on other host species, wheat and barley grass, as distinct formae speciales. Wheat stripe rust, P. striiformis f.sp. tritici has a low level of infection on barley and does not cause significant damage to barley crops. Susceptible barley cultivars lose approximately 10% yield due to barley grass stripe rust (Wellings et al., 2000). Isolates of the stripe rust pathogen, which demonstrated adaptation to cultivated barley, were described by European workers in the late nineteenth century (Line, 2002). Barley stripe rust has caused significant problems in winter barley production in Europe, the UK and the Netherlands since the 1960s (Stubbs, 1985), Colombia and South America from 1975 (Dubin & Stubbs, 1986), Mexico from 1990 and the USA from 1991 (Marshall & Sutton, 1995). Since 1991, stripe rust of barley has quickly spread and become established in the south-central and western USA and is now the most important disease of barley in western the United States (Line, 2002; Chen, 2004). When field testing of Australian barley commenced at the International Centre for Wheat and Maize Improvement (CIMMYT), Mexico, more than 80% of current varieties were very susceptible to barley stripe rust (Wellings & Park, 2003). With offshore testing and the availability of molecular markers to select resistance genes (Cakir et al., 2003) pre-emptive breeding is being initiated to give Australian barley varieties protection from stripe rust incursions. Description P. striiformis is a hemiform rust with uredinial and telial stages being produced during its life cycle. The urediniospores complete multiple asexual cycles throughout the growing season and these cycles cause the principle damage to cereal crops (Figure 1). Spores are binucleate. Infection hyphae have 4 nuclei (Line, 2002). Telia, producing teliospores, form on heavily infected leaves and leaf bases at the end of the season. The urediniospores are yellow to orange in colour, spherical, echinulate and 28-34 m in diameter (Singh et al., 2002). Spores infect leaves and spikelets and develop sporulating pustules in rows of varying lengths giving the appearance of narrow yellow stripes. Striping is not evident on seedling leaves, rather the infection covers the leaves in a random fashion. The fungus may affect leaf sheaths and heads in heavy epidemics (Adams, 1997; Bariana, 2004). The rust is an obligate pathogen and therefore, must reside within a living host for survival (Adams, 1997). Conditions are suitable for rust development between April and December and in most years infections can be observed in crops by September. 6 Figure 1. Life cycle of Puccinia striiformis. 1.1 Spread Urediniospores of barley stripe rust are capable of moving great distances on wind currents. Barley stripe rust in South America migrated from Colombia to Chile over a period of only a few years as a result of wind dispersal (Dubin & Stubbs, 1986). Intercontinental air travel from Europe has been predicted to be the pathway pattern of P.s. hordei on barley in Columbia. 1.3 Strains Using a set of 11 differential barley genotypes, 69 races of barley stripe rust have been identified as occurring in the United States (Chen, 2004). Since 1998 certain races have become predominant but because of non race-specific resistance, selection pressure has been low and the rust population still consists of numerous races (Chen, 2004). In Europe, there has been less race diversity identified with race 24 being predominant (Stubbs, 1985; Dubin & Stubbs, 1986). 1.4 Hosts Spring barley, Hordeum vulgare, is the primary host for the disease. Certain races of the rust will also survive on wild barley species such as H. jubatum (foxtail barley) and H. leporinum (barley grass)(Marshall & Sutton, 1995). 7 1.5 Losses Resistant varieties show no significant yield loss. Yield losses of 30-70% were estimated when barley stripe rust was first identified in South America (Dubin & Stubbs, 1986). In 1995, the highest grain yield loss in susceptible US varieties was 72% (Marshall & Sutton, 1995). Recently, commercially grown susceptible US cultivars showed a 20% yield loss and cultivars with moderate levels of adult plant resistance showed a 12% yield loss. (Chen, 2004). In recent years, US state- wide losses have been lower as highly susceptible cultivars are rarely grown. However, severe barely stripe rust still appears in test plots on susceptible lines and is a continuing threat (Jackson. 2003). 1.6 Control There are several control options available to producers: 1. Seed treatment with fungicides may delay the onset of an epidemic by preventing early build up of the disease on seedlings (Brown et al., 2001).