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The “Rice diseases workshop: improving epidemiology and disease diagnosis for sustainable management” was organized by AfricaRice, CIRAD and IRD with the financial support of Global Rice Science Partnership (GRiSP). It took place in Montpellier (France), from 24 to 26 November 2014.

Rationale

Rice is one of the major staple food in the world and a pillar for food security in many developing countries. However, biotic stresses constantly threaten sustainable production of rice due to their dramatic impact on grain yield and quality. As a prerequisite for efficient and innovative control strategies, it is mandatory to improve the sharing of information among experts in a global and regional scale and increase expertise capacities in epidemiology and disease diagnosis.

While identification of some resistance genes is addressed within the theme 1 and plant breeding issues are the focus of the theme 2, much less is dedicated in GRiSP product lines on environmental and epidemiology aspects of rice pathology.

The objective of the workshop was to fill this gap: how can environmental, agro-ecological conditions and rice growing systems modulate the pathogen population diversity, and finally hamper the effectiveness of durable resistance deployment? How can the community of rice pathologists set an organization to monitor long-term observations of rice-pathogens interactions and epidemiology in representative rice growing conditions?

In some regions, and particularly in Africa, plant pathologists are few compared to the number of diseases rice is facing and financial resources devoted to research are limited. In this context, it is critical to set up networks of specialists to be able to share experience and to improve coordination. The workshop aimed at contributing to strengthening and extending partnership and building capacities in rice pathology.

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Objectives

The main content of this workshop were twofold: i) initiate the elaboration of robust, cost-effective and transportable epidemio-survey and diagnosis protocols and ii) design the framework of an international network of rice pathologists taking in consideration continental/regional specificities (i.e. mainly targeted to Africa).

The workshop was also an opportunity to review results and progress of the current GRiSP   MENERGEP project (“Methodologies and new resources for genotyping and phenotyping of African rice species and their pathogens for developing strategic disease resistance breeding programs) to feed the discussions.  

The first objective was to create a dynamic for the elaboration and implementation of standardized protocols for sampling, pathogen strain isolation, storage and molecular or phenotypic characterization, and diagnosis. The workshop also envisaged how practical training to these methods can be organized for NARS (National Agricultural Research Systems) students and how they can be supported. The discussions particularly focused on :

• Standardized protocols for main rice disease diagnosis, genotyping and phenotyping;

• Pathogen isolate collections: rules, management and back-up strategies;

• Pathogens epidemio-survey: a multi-pathogen approach and better connection with breeding taskforce and rice-Hubs in Africa;

• Emerging diseases: symptoms, distribution and diagnosis tools.

The final goal of the workshop was also to elaborate on the creation of a network of rice pathologists, which could ensure in the future the structuration of a trans-disciplinary community of scientists aiming at taking forward and sustaining the actions anticipated during the workshop. Notably, several initiatives are already in progress and one objective was to evaluate how these initiatives can be federated.

Organization of the meeting and outputs

We gathered a group of 30 experts including 10 reference persons for each of the main rice diseases in Asia, Africa and South-America, 12 NARS scientists working in Africa on rice diseases and 8 scientists from CIRAD and IRD (see list of participants in annex 1). During the first day, the international experts were invited to give a presentation presenting the main rice diseases and the current state of the art of research on these diseases (see program in annex 2 and abstracts in annex 3). This first day was opened to the public and attracted ca 50 additional participants. This action contributed to show to the local scientific community 1) the importance of rice diseases worldwide, 2) the quality of research done on this theme, and 3) the task force on rice diseases in Montpellier.

The second day and third day were restricted to the 30 experts. The first half of the second day was dedicated to exchange of information by a brief self-presentation of each

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participant followed by selected presentations of international and national on-going projects on rice diseases in Africa (see program). The second half of the first day was organized as round tables on four topics: 1) Epidemiology and forecasting, 2) Sampling, isolating and storing pathogens, 3) Genotyping pathogens and 4) Phenotyping pathogens. The main outcomes of these round-tables are presented page 4 and more details are provided in annex 4 to 7. Finally, the last morning was dedicated to a general discussion on the outputs of the workshop with special emphasis on the future of research on rice diseases in Africa and on the actions to promote an African Network for rice diseases. This session leaded to recommendations and initial actions which are given page 6 and in annex 8.  

 

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ROUND-TABLES REPORT

On the second day of the workshop, participants were split into four subgroups (7-8

scientists per group) to initiate reflection on important topics for rice pathology. The four selected themes were: (1) Epidemiology and forecasting, (2) Sampling, isolating, storing pathogens, (3) Genotyping pathogens, (4) Phenotyping pathogens. The brainstorming focused on the following questions: objectives (what for?), methodology (how?), problems (how to improve?) and perspectives (what’s next?). A first hour was dedicated to discussion inside the subgroups, and was followed by 20 minutes restitution on each topic in plenary session to open the discussion with all the participants. Each group was composed of scientists from different countries and specialists of different diseases, which allowed fruitful discussions, as insights from practices on one disease help in improving research on the other diseases

Detail reports of each sub-group are available in Annex 4 to 7, but the main conclusions may be emphasized. First, as underlined by the first sub-group, epidemiological field survey can yield a wealth of information on disease transmission and environmental preferences and are also necessary to justify the effort invested in control strategies. Such data are however drastically lacking in Africa, except partially for the major diseases (blast and RYMV). Methodological innovation is needed to improve diagnosis in the field and/or in the lab, as symptom-based diagnosis, whether frequently used, is often insufficient, especially in case of multiple infections. These new methodologies have to be applicable in the African context (robust and cost-effective methods). Finally, the establishment of standardized protocols appears to be a critical point to get a comprehensive picture of rice disease epidemiology at the continent level.

The sampling and storing of pathogens appeared to be a key point to get a comprehensive picture of population diversity in order to understand pathogen evolution and evaluate control strategies. The minimum requirement is often the maintenance of cold chain from the field to the lab long-term storing, but a good symptom recognition ability, that may requires capacity building, is also necessary. Among the methodologies identified to improve the sampling, isolation and storing of pathogens, the participants mentioned the standardization of protocols and passport data, as well as duplication of stored samples, if possible, in Africa and Europe, which both may involve a network of pathologists. Besides, epidemiological surveys and pathogen sampling targeting various diseases at the same time (multi-pathogen strategy) should be favored as 1) it is an efficient and cost-effective way to bring new insight for each disease and 2) important patterns could emerge from the joint analysis of different diseases as the pathogens may influence each other (multiple infection patterns).

Genotyping of the pathogen is essential both at the strain level, to help diagnosis, evaluate the diversity or manage the collections, and at the population level, to characterize

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their structure and their dynamics in different ecosystems. Genotyping is also a tool for the identification and characterization of pathogenicity and virulence factors. A good knowledge on these different points is finally a key element for the deployment of sustainable resistances. Genotyping mainly relies on molecular tools and different type of PCR-based markers. Accessibility to those technologies constitutes a limit for some labs in Africa. This constraint may be solved by the improvement of labs capability, access to platform or the development of more robust and cheaper methodologies. Once again, the standardization of protocols will contribute to a more efficient pathogen genotyping.

The phenotyping of pathogens will give a comprehensive picture of the virulence spectrum of pathogen populations and provide tester strain and is thus directly associated to the breeding for resistance. The two main requirements for pathogen phenotyping are standard inoculation and disease assessment protocols and the availability of differential rice accessions, near-isogenic lines being the best material. Such protocols or materials are partly available for the main diseases (blast, BLB, partly for RYMV) but have to be completed and improved. To this purpose, a coordination network would optimize the effort required for the development of near-isogenic lines in adapted genetic backgrounds and for the seeds multiplication and distribution.

Finally, despite the specific focuses of each sub-group, a general consensus emerged on the need to share methodological knowledge with common protocols, and biological material, such as tester accessions, and eventually duplication of pathogen collections. Such common practices are differently advanced for the different diseases, blast being probably the disease for which the more standardized procedures are available, but have to be generalized. In this context, a functional network of rice pathologists would contribute to 1) share methodologies through available standardized protocols and 2) establish pathogen collections and differential lines for main rice diseases in Africa.

 

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AN AFRICAN NETWORK FOR RICE DISEASES

The idea of creating an African network for rice diseases was raised as a major priority by a set of rice pathologists, who gathered in the frame of a working group held at the last African Rice Congress in Yaoundé (Cameroon) in October 2013. As a main statement they recognized that the number of rice diseases experts are insufficient in Africa, while equipment and facilities are also limited throughout the continent. It was highlighted a crucial need to optimize their use and link available rice pathologists through community building in order to optimize R4D activities, avoid duplication and mobilize adequate funding.

On the strength of these statements, we seized the opportunity to formalize the establishment of a pan African network for rice diseases, having close at hand a representative albeit not complete subset of scientific actors highly involved in the field, namely rice pathologists from diverse national agricultural research and extension systems (NARES) in Africa, international research centers (AfricaRice, CIAT, IRRI, IITA), Universities, and partner institutions (IRD, Cirad).

As collectively stated, the main objectives of the network will be to ease the structuration of a community in improving its visibility, fostering capacity building, seeking funding, avoiding redundancy and stimulating complementary collaborative research projects.

Backed by AfricaRice, the network which is collectively coordinated by Drissa Silué (ARice, Benin), Joseph Birigimana (IRRI, Burundi) and Issa Wonni (INERA, Burkina Faso), will be organized around six main areas (detailed in annex 8) :

• Communication

• Funding of network operating costs

• Establishment of standardized protocols

• Capacity building / training

• Network expansion

• Research activities

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ANNEX 1: LIST OF PARTICIPANTS

Name Surname Institute Email

Adamou Basso INRAN, Niger adamoubasso@yahoo.fr

Albar Laurence IRD, France laurence.albar@ird.fr

Anthony François IRD, France francois.anthony@ird.fr

Baimey Hugues IITA, Benin baimeyhugues@gmail.com

Bigirimana Joseph IRRI, Burundi j.bigirimana@irri.org

Bouet Alphonse CNRA, Côte d'Ivoire bouetalph@gmail.com

Choi Il-Ryong IRRI, Philippines i.choi@irri.org

Correll Jim U. ARK, USA jcorrell@uark.edu

Cruz Nollie IRRI, Philippines c.veracruz@cgiar.org

De Waele Dirk IRRI, Uni., Belgium dirkdewaele@pandora.be

Fukuta Yoshimichi JIRCAS, Japan zen@JIRCAS.AFFRC.GO.JP

Galzi Agnes IRD, France agnes.galzi@ird.fr

Ghesquière Alain IRD, France alain.ghesquiere@ird.fr

Harinjaka Raveloson Fofifa, Madagascar raveloharinjaka@yahoo.fr

Hébrard Eugénie IRD, France eugenie.hebrard@ird.fr

Kossi Kpemoua ITRA, Togo kossi.kpemoua@gmail.com

Kouassi Nazaire CNRA, Côte d'Ivoire kouassinazaire@yahoo.fr

Kumashiro Takashi AfricaRice, Benin T.Kumashiro@cgiar.org

Leach Jan CSU, USA Jan.Leach@ColoState.EDU

Mosquera Gloria CIAT, Colombia G.M.Mosquera@cgiar.org

Ndikumana Innocent RAB, Rwanda ndikin@yahoo.fr

Oliva Ricardo IRRI, Philippines r.oliva@irri.org

Ouegraogo Leonard INERA, Burkina Faso tinkoglega@gmail.com

Rakotomalala Mbolarinosy Fofifa, Madagascar mbolarinosy@yahoo.fr

Sester Mathilde CIRAD, Madagascar mathilde.sester@cirad.fr

Silué Drissa AfricaRice, Benin D.Silue@cgiar.org

Szurek Boris IRD, France boris.szurek@ird.fr

Tharreau Didier CIRAD, France tharreau@cirad.fr

Tollenaere Charlotte IRD, France charlotte.tollenaere@ird.fr

Traoré Edgar INERA, Burkina Faso traorevalentin@yahoo.fr

Wonni Issa INERA, Burkina Faso wonniissa@yahoo.fr

Zhou Bo IRRI, Philippines b.zhou@irri.org

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ANNEX 2: WORKSHOP PROGRAM  

Monday November 24 (open to the community)

A. Ghesquière (IRD, France): Workshop introduction Session 1: Rice diseases worldwide G. Mosquera (CIAT, Colombia): Rice diseases in Latin America N. Vera-Cruz (IRRI, Philippines): Rice diseases in Asia D. Silué (AfricaRice, Benin) and J. Bigirimana (IRRI, Burundi): Rice diseases in Africa Session 2: Research on rice pathogens and control strategies I.R. Choi (IRRI, Philippines): Introduction to viral diseases and control methods J. Leach (CSU, USA): Introduction to bacterial diseases due to Xanthomonas and control strategies R. Oliva (IRRI, Philippines): Tapping into rice associated microbes: Pseudomonas fuscovaginae J. Correll (UARK, USA): Introduction to rice blast and control methods B. Zhou (IRRI, Philippines): Rice - rice blast molecular interactions Y. Fukuta (JIRCAS, Japan): Phenotypic characterization of rice - rice blast interactions Tuesday November 25 (restricted)

1. Short self oral presentation of participants 2. Presentation of projects on rice diseases in Africa:

A. Ghesquière & H. Baimey: Menergep (GRiSP New Frontier project) J. Corell: BBSRC – Rice Blast Project C. Tollenaere & E.Traoré: LMI PathoBios (INERA-IRD) J. Bigirimana: Work on rice diseases at IRRI East Africa T. Kumashiro: Breeding strategies at AfricaRice A. Bouet & N. Kouassi: Ongoing and future projects in Ivory Coast B. Adamou: Ongoing and future projects in Niger K. Kpemoua: Ongoing and future projects in Togo I. Nkikumanab: Ongoing and future projects in Rwanda M. Rakotomalala & H. Raveloson: Ongoing and future projects in Madagascar

3. Brainstorming on four topics: 1) Epidemiology 2) Sampling, isolating and storing pathogens 3) Genotyping pathogens 4) Phenotyping pathogens.

Wednesday November 26 (restricted)

General discussion on the actions to promote an African Network for rice diseases and recommendations for future research on rice diseases in Africa.

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ANNEX 3: ABSTRACT OF PRESENTATIONS

G. Mosquera (CIAT, Colombia): Rice diseases in Latin America CIAT’s Rice Breeding program is continuously working to improve productivity in Latin America and The Caribbean (LAC) region. Among the main challenges for rice producers in LAC in terms of diseases are Rice Blast and grain sterility complex. Rice pathology at CIAT is mainly focused in studying these two limitations where pathogen population structure and R genes performance are the main topics for blast, and diagnostic, pathogenicity tests and germplasm screening being the main research targets for B. glumae one of the agents involved in grain sterility complex.

So far we have identified nearly 100 Blast races in Colombia and the most aggressive ones have been found in the “hotspot” Santa Rosa, where breeders select for disease resistance under natural infections. On the other hand we have identified protocols for sampling, diagnosis, bacteria isolation, and testing pathogenicity in B. glumae under greenhouse conditions. Additionally reproducible methods are being used to screen germplasm to identify sources of resistance for B. glumae not only under greenhouse conditions but also under natural infections in a “hotspot”.

Results obtained so far demonstrated that most known R genes for blast are defeated by the fungus in Colombia, specifically in the selection site of CIAT Rice breeding program, what requires the identification of new resistances genes for the breeding program since at least in Colombia the pathogen is highly diverse at pathogenic level. Information about this condition in other LAC regions is unknown but breeders should be prepared in advance assuming fast pathogen dispersal inside the region. For B. glumae, preliminary results have shown potential sources of resistance to this disease with good correlation between greenhouse and field evaluations. Genetic studies will be carryout soon to identify the location of the genes involved in this trait.

The main focus of our research in the future to support Rice production in LAC is to identify new sources of resistance for Blast to assure good disease control using resistant varieties and to identify the genes involved in resistance to B. glumae and their molecular markers to be transferred to the elite lines in development.

D. Silué (AfricaRice, Benin) and J. Bigirimana (IRRI, Burundi): Rice diseases in Africa Rice has become a very important crop for Africa as most countries cannot meet their local constantly growing demand for use as food and rely on increasing importations to feed their populations. This affects food security and many countries like Nigeria have taken steps to increase production and meet half of the demand by 2020. This will partly be done by increasing yield which is still very low in average (1.0 to 1.5 t/ha) as compared to Asian countries where yield average is over 5 t/ha. Among the many factors limiting rice production in Africa, rice diseases are major constraints and IRRI and AfricaRice are putting emphasis on improving resistance of farmer adopted varieties and also developing improved lines with stable and durable disease resistance. Rice blast (Magnaporthe oryzae), Rice Yellow Mottle

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Virus (RYMV), bacterial leaf blight (bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae and bacterial leaf streak by X. oryzae pv. oryzicola) are among the most important diseases. As emerging, cited diseases are brown spot (Cochliobolus miyabeanus), narrow brown spot (Sphaerulina oryzina) and Rice Stripe Necrosis Virus or RSNV. New bacterial blights which are wide spread (e.g. Pantoea spp.) are being reported by AfricaRice. The importance, distribution and ecologies of these diseases are presented in the present presentation.

I.R. Choi (IRRI, Philippines): Introduction to viral diseases and control methods At least 16 virus species have been reported to cause yield losses of rice cultivated worldwide. A majority of rice viruses are transmitted by leafhoppers or planthoppers, and are found in Asia. Besides the rice viruses in Asia, Rice yellow mottle virus (RYMV) in Africa, and Rice hoja blanca virus (RHBV) in South America have been recognized as a serious threat to rice production in the respective continents. Various efforts were already made to manage rice virus diseases in fields. Control measures for rice viruses diseases are largely divided into the resource management and the genetic approaches. The resource management approach aims at prevention of rice virus diseases by reducing virus sources and virus-transmitting insects, and by avoiding virus disease spread. The reduction in virus sources can be achieved by simple practices such as eradication of infected plants and ploughing of affected fields. Application of insecticides is considered as an acute measure to reduce the virus-transmitting insects when sudden outbreaks of rice virus diseases occur. Light trap devices can also serve as a routine measure to reduce the level of virus-transmitting insects in fields. The avoidance of virus disease spread can be achieved by adjusting initiation timing of rice cultivation through careful monitoring for migration of insect populations. The genetic approach to control rice virus diseases includes deployment of resistance genes to rice viruses and those to virus-transmitting insects. Genes controlling resistance against RYMV, Rice strip virus (RSV), and Rice tungro spherical virus (RTSV) are characterized well, and have been introduced into various rice varieties though DNA marker-assisted selection. The presence of resistance genes against RHBV and Rice tungro bacilliform virus (RTBV) were also reported. Besides natural resistance to the rice viruses, many attempts have been made to develop artificial resistance traits against viruses such as RSV, RYMV, and RTBV using novel genetic engineering technologies. Several dozens of genes associated with resistance to virus-transmitting insects, such as brown planthoppers, small brown planthoppers, and green leafhoppers, have been reported. Such insect resistance genes have been widely exploited to manage the level of rice virus diseases in fields, although the effectiveness of insect resistance genes is considered to be limited due to quick adoption of the insects to rice varieties with particular insect resistance genes. Combination of the resource management and the genetic approaches was proven to be effective to reduce the occurrences of rice virus disease in fields, however, accurate identification of rice viruses and outreach programs to deliver such control measures should be accompanied for sustainable management of rice virus diseases.

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J. Leach (CSU, USA): Introduction to bacterial diseases due to Xanthomonas and control strategies Bacterial blight (BB) and bacterial leaf streak (BLS) are the two most important bacterial diseases of rice worldwide. These diseases are caused by two pathovars of Xanthomonas oryzae: X. oryzae pv. oryzae (Xoo) that causes BB, and X. oryzae pv. oryzicola (Xoc) that causes BLS. Xoo and Xoc are not found in the USA. Xoo and Xoc are widely distributed and endemic in many countries in Asia, Africa and Australia, but they have not been found in North America. A third group of X. oryzae is found in rice-producing states within the USA, and this group causes very weak disease symptoms that resemble BB. The USA X. oryzae, which has no pathovar designation, is genetically distinct from Xoo and Xoc, and contains none of the Transcription activator-like (TAL) effectors that are common to Xoo and Xoc. Because they are TAL-deficient, these strains of X. oryzae provide a platform for investigating the biological roles of TAL effectors as well as a tool for identifying novel sources of resistance to BB and BLS.

Control of BB and BLS are typically through genetic resistance. BB is most effectively controlled through the use of qualitative resistance governed by single resistance (R) genes. Changes in the race structure of Xoo populations can render R genes ineffective, so there are continual efforts to identify new sources of resistance, including sources of quantitative resistance. To date, the sources of qualitative resistance for BLS are very limited, and no race structure for Xoc has been reported. Thus, most sources of resistance for BLS are quantitative. Novel rice genetic populations, such as MAGIC (multiparent advanced generation intercross) populations are being screened for new sources of resistance to both BB and BLS.

Using comparative genomic approaches, diagnostic primers were developed that distinguish Xoo, Xoc, and the USA X. oryzae from one another by PCR-based approaches. With these primers as a foundation, improved protocols, such as Loop-Mediated Isothermal Amplification (LAMP), that allow for detection of the pathogens in seed- and plant-tissue in the field, are being evaluated internationally.

J. Correll (UARK, USA): Introduction to rice blast and control methods Rice blast disease, caused by Magnaporthe oryzae, is a major constraint to rice production in most rice productions areas, including the Southeast U.S. Arkansas is the largest rice producing state in the U.S., growing approximately 50% of the acreage. Although rice blast epidemics have been sporadic, highly dependent on environmental conditions, severe epidemics were observed in Arkansas in 1992, 1994, 1996, 1999, 2004, and 2009. Neck and panicle blast is the most predominant symptom and a significant impact on yield was observed in the years with severe rice blast. Isolates of M. oryzae collected over the past 20 years belong to one of four distinct genetic distinct lineages that have been found in the contemporary population in Arkansas. However, there is considerable virulence diversity within the four distinct lineages. To evaluate the effectiveness of resistance genes, a collection of U.S. isolates of M. oryzae were evaluated for virulence using 25 to 40 U.S. rice differentials and two sets of differentials developed by IRRI. The first set comprised 31

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monogenic lines with 24 resistance genes and, the second set had 20 lines with 14 resistance genes. In addition, four NERICA lines were evaluated. A total of 12 different U.S. reference races and a collection of field isolates representative of the genetic diversity in the U.S. population were evaluated on the rice lines. The sets of IRRI differential lines discriminated the isolates into groups that differed from groupings based on the U.S. differentials. Two NERICA lines were resistant to all the isolates that were evaluated. NERICA lines did not help in virulence discrimination among the isolates evaluated. However, the resistance genes in these lines could be exploited as potential new sources of resistance to rice blast in the U.S. Future efforts continue to focus on phenotyping additional field isolates to fully characterize isolates which can overcome specific resistance genes.

B. Zhou (IRRI, Philippines): Rice - rice blast molecular interactions The rice blast disease caused by the fungal pathogen Magnaporthe oryzae poses a constant threat to the stable rice production. Utilization of host resistance (R) genes constitutes the most sustainable and economical means to tackle this devastating disease. However, most R genes are prone to collapse in several years after deployment due to the extreme variability of pathogen. Moreover, R genes usually work differentially in different growing areas by matching different pathogen population. Therefore, the understanding of interaction between rice and rice blast becomes prerequisite for developing a sustainable strategy for the controlling of rice blast disease. In this presentation, the progress on the characterization of resistance genes in rice and avirulence genes in rice blast will be updated. The studies of two pairs of R/Avr genes, i.e., Pi9/AvrPi9 and Piz-t/AvrPiz-t, will be presented as examples for demonstrating the different mechanisms underlying the recognition between different R and Avr genes. Finally, pathogen informed R gene deployment scheme will be discussed. The Avr gene based diagnosis tool developed for monitoring the dynamics of rice blast pathogen populations will be described and discussed.

Y. Fukuta (JIRCAS, Japan): Phenotypic characterization of rice - rice blast interactions Differential system consists of (1) a differential varieties’ set which has different resistance genes in each genetic background and (2) a standard differential blast isolates’ set which clarifies the pathogenicity, is a basic tool for blast study and breeding. Unfortunately, it has yet to been developed and used in developing countries in tropical regions.

JIRCAS, together with IRRI, have developed the international differential varieties’ sets by backcross breeding with some susceptible Chinese variety, Lijiangxintuanheigu (LTH, Japonica-type), CO39 (Indica-type), and US-2 (Indica-type), with each variety introduced with a different resistance gene in the genetic background.

In cooperation with countries in Southeast South Asia, and Africa, an international collaborative research on blast disease is being conducted using these international differential varieties developed by JIRCAS and IRRI. These studies aim to create a durable protection system against blast disease through the development and application of the differential system.

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The network studies used (1) a common criteria for evaluating the virulence of blast isolates to each differential variety and (2) a designation method for blast isolates based on the reaction patterns to differential varieties’ set as race, allowing all data to be shared and compared among the participants. As a result, (3) the diversity and distribution of blast races at regional and global levels were clarified, and (4) the use of international standard differential blast isolates (ISDBIs) made possible the characterization of resistance genes and the evaluation of resistance in rice varieties. In the breeding studies, (5) the genetic variation of resistance in rice germplasm, and (6) the clarification of genotypes of resistance genes of leading varieties in each country were carried out.

Some research findings for the global differentiation of blast races and genetic variation of rice germplasm under the collaboration will be shown, and then a candidate set of international standard differential blast isolates will be introduces.

C. M. Vera Cruz (IRRI, Philippines): Rice Diseases in Asia The rice production environments in Asia, especially in the tropics, are habitats of many rice pathogens causing varying degrees of damage. Quantified annual yield losses based on surveys due to a combination of rice diseases ranged from 1 to 10%, whereas Somerville & Briscoe (2001, Science) noted that “up to 40% of plant productivity in Africa and Asia, and about 20% in the developed world, is lost to pests and pathogens”. In the rainfed and irrigated rice environments where 85% of the world’s rice output is coming from, diseases cause yield losses ranging from 50-86 million tons yearly. They are further exacerbated by rapidly warming climate, where higher growing temperatures impact agricultural productivity and food security. As such, models of plant disease have now been developed which incorporated more sophisticated climate predictions. Generally in Asia, 10 bacterial, 29 fungal, and 10 viral diseases affecting seedlings to grains have been reported so far. Of these, the current main yield reducing diseases are sheath blight (caused by Rhizoctonia solani) > brown spot (Bipolaris oryzae) > bacterial blight Xanthomonas oryzae pv. oryzae), blast (Magnaporthe oryzae), viruses. Recently, emerging diseases such as false smut (Ustilaginoidea virens), bacterial leaf streak (Xanthomonas oryzae pv. oryzicola) and grain diseases caused by a complex of bacterial and fungal diseases, of which many are seed-borne, have also been reported in China, India and other parts of Asia. Other emerging diseases commonly reported in water scarce environment and where aerobic rice system is practiced are root diseases caused by root rotting Pythium arrhenomanes, Pythium graminicola and parasitic nematodes (root knot nematodes, Meloidogyne graminicola). Disease control using chemicals is commonly practiced in the temperate or subtropical production environments where farmers apply fungicides for controlling rice blast and sheath blight. However, despite regional differences in control methods, planting resistant varieties is considered most effective, particularly for bacterial blight and blast. Limitations however are recognized in using resistant varieties alone to manage diseases due to pathogen variation. Thus, at IRRI, we are revisiting the populations of these pathogens using ‘hotspot strategy’ in combination with monitoring beyond hotspots when diseases are endemic in a region.

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Knowledge of pathogen population biology and understanding its population structure are important components in breeding for resistance to such diseases. In general, combinations of disease management strategies that include both resistance gene-based and resource-based approaches such as fertilizer and water management, field sanitation, fungicide application (to a limited extent especially for resource-poor farmers), synchronous planting, crop rotation in rainfed environment, and seed health management (especially for seed-borne diseases) have also been practiced by farmers.

R. Oliva (IRRI, Philippines): Tapping into rice associated microbes: Pseudomonas fuscovaginae Seed-borne and seed-transmitted pathogens, such as Pseudomonas fuscovaginae, cause direct economic losses to farmers and represent an important obstacle for germplams exchange and quarantine measures. Although the pathogen infects important crop species such as maize, sorghum, wheat, and rice, very little is known about genomic features related to its life cycle. In this study, we analyzed the whole genome sequence of P. fuscovaginae strains isolated from rice in different parts of the world and compare its functional annotations with other plant pathogenic Pseudomonas genomes. In addition, we studied transcript accumulation of bacteria genes during its interaction with rice and provide evidence of a consorted array of mechanism used by the pathogen to colonize plant tissue. Comparative genomics analysis of P. fuscovaginae ecotypes reveals structural polymorphism and high levels of genetic diversity. We also compared the core genome of P. fuscovaginae with all Pseudomonas genomes reported so far and identified unique set of genes. P. fuscovaginae genome harbors clusters of pathogenicity-related genes, several secretion systems, toxin and a range of secondary metabolites that appear to facilitate infection of rice cell. Compare to other pathogenic bacteria that causes brown rot, cell-wall degrading enzymes are poorly represented in the P. fuscovaginae genome. Our data will contribute to the understanding of the molecular pathogenesis and natural variation of rice bacterial sheath brown rot. Transcriptome profiling of rice genes is underway and will provide additional clues of pathogenicity mechanism reflected on rice immune response.

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ANNEX 4: EPIDEMIOLOGY AND FORECASTING

Participants: S. Bellafiore, A. Bouet, J. Corell, J. Leach, H. Raveloson, M. Rakotomalala, C. Tollenaere The participants first realized the need to agree on the definition of the term “epidemiology” and what would be discussed during the round table. Epidemiology is the Characterization of spatio-temporal disease dynamics, and the investigation of different biotic and abiotic drivers (Basically: Who is there? Where and when? Why?). Diagnostic is needed in this context as a tool. Incidence (proportion of infected plants in a field and proportion of infected fields at the regional scale) has be taken into account as well as severity: leaf area covered, amount of plant destroyed by the disease, yield quantity and quality decrease... for the question: Is this disease important? What for?

- Better understanding of the disease (vector, transmission modes, refugees, etc…) - Feed predictive models - Early documentation of emergence - Justify the funding and effort invested (in resistance breeding for example)

Importance of threshold to apply control diseases: Should it be controlled ? If the answer is yes, how to design efficient control strategies: chemical (useless chemical spray?), agronomical practises, resistance breeding (adjusted effort on most important diseases) How? Diagnosis: in the field (symptoms)/ in the lab

Pathogen Methods

Viruses RYMV symptoms sometimes clear but expertise needed and depends on varieties, ELISA is needed for confirmation

Bacteria Great need for molecular methods for BB/ BB-like symptoms, BLS symptoms are clearer, but many bacteria hard to distinguish are emerging

Fungi Expertise is needed to identify the symptoms different fungi (blast, brown spot); microscopy can help

Root-knot nematodes

Abiotic stresses (nutrient starvation, water loss) can give similar symptoms, checking root system and examining galls under microscope is needed, molecular markers are needed to identify the species

General remark: multiple infections may greatly complicate symptom-based diagnosis…

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Survey protocols: - Disease identification and quantification: How to quantify the disease ? Standardized survey protocols needed to obtain representative data - Spatial context of study: Appropriate spatial scale, GIS - Factors: What kind of environmental factors have to be studied? Study involving alternative hosts, vectors, soil, water... ?

Survey methodology will depend on the question addressed, but standardization of the protocols between researchers is needed to get a comprehensive picture at the continental level.

How to improve? To date, few epidemiological studies of rice diseases in Africa, except RYMV and blast in Madagascar (but many questions remain as well). Very little work on bacteria, nematodes…

What’s next? Training/expertise is needed for researchers, and increasing farme's knowledge is important as well Need to share informations (documenting emergence for example) though the network Toward high-thoughput epidemiological studies (remote sensing, drones??) of rice diseases in Africa

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ANNEX 5: SAMPLING, ISOLATING AND STORING PATHOGENS

Participants: H. Baimey, G. Mosquera, J. Bigirimana, A. Galzi, I. Ndikumana, D. Silué, M. Sester, B. Szurek What for?

• detect the presence of disease

• evaluate the distribution of the disease • analyse the population diversity and evolution

• confirm the presence of the disease

• test alternative hosts • monitor the durability of the resistant genes

• test the effectiveness of control strategies

• establish reference collections • standardize the protocols

How?

Pathogen Methods

Viruses Symptomatic leaves, Gloves/disinfection, Cool box if possible if the virus is not stable conservation best at – 20°C under vacuum and possible as dried leaves Isolation is done by propagation( by mechanical inoculation or use of vectors) Keep always small original sample

Bacteria Symptomatic tissues, Cool box if long trip conservation best at – 20°C Wash leaves with 70% Ethanol, isolation by grinding tissues and serial dilution Sub culturing of pure culture

Fungi Symptomatic tissues, paper bags conservation best at – 20°C Wash leaves with Ethanol, humid chamber of affected tissues Single spore/Monohyphal, isolation and propagation Grow the isolates on filter paper, dry them and store -20 c

Root-knot Symptomatic tissues and soil

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nematodes Keep the samples in cool box Select the appropriate extraction method Store the nematodes at 10°C or -20°C

Multiply on susceptible host  

Passport data: collection date collector name GPS/Location name of variety ecology crop management(fertilization, rotation,,,,,) organ (leaf, roots, stems,soils,,,) sample nomenclature other symptoms incidence

How to improve? Potential problems Solutions

symptoms recognition Capacity building

multiple infection Capacity building Multiplex diagnosis

deterioration of samples Isolation in situ Field labs Duplication of samples Develop simple protocols

Conservation conditions Reference storages in Africa and Europa

phytosanitary requirements Good planning Isolation on site

impact of sampling on the surveyed fields Use of drones  

What’s next? • Regular samplings on same site • Unified database (storage information) • Adapted sampling strategies • Multi pathogens samplings

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ANNEX 6: GENOTYPING PATHOGENS

Participants: B. Zhou, R. Oliva, F. Anthony, K. Kossi, I.R. Choi, L. Ouedraogo, D. Tharreau, E. Hébrard. What for?

• To help the diagnosis, to design efficient tools • To manage pathogen collections (redondancy) • To determine the ID of reference strains • To characterize the pathogen diversity

• To describe the structure of the pathogen populations (tool for epidemiology) • To understand evolution of diseases and pathogens (emergence/ re-emergence) • To monitor dynamic and structure of populations in differents ecosystems

• To identify determinants of the pathogenicity (gene discovery) • To investigate resistance /virulence mechanisms

• To identify gene constraints and evolutionary potential • To predict pathogenicity / virulence and pathogen evolution • To monitor the stability/instability and the rate of change • To generate data in a view to resistance durability • To deploy resistant varieties taking into account diversity and adaptability

How? Pathogen Methods

Viruses

Serology/ELISA RT-PCR ( ex: CP) Direct sequencing / Full-length

Bacteria (different species)

Multiplex PCR, VNTR (low scale) SNPs (high scale) Direct sequencing

Fungi (=Blast?)

Microsatellites Avirulence genes ITS sequences SNPs

Nematodes (different species)

Mitochondrial SSCR

SNPs  

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How to improve? • serological sensitivity not enough but cheap • PCR contaminations • Pb if new/unknow viruses (metagenomics) • multiplex-PCR depends on the species of bacteria for ex • Rate of pathogen evolution (incidence on molecular tools) • Correlation between genotype/pathotype (pb avr genes) • Informative/neutral markers • Access in Africa (platform?), access of products (liquid nitrogen,…) • Capability of labs, heteregenous • Pb of technology evolution • Unique Chips for all the rice pathogens (leaf/root) • Epigenetics ?

What’s next?

• to establish cheap, efficient and precise genotype approaches • adapted to African countries • directly linked to virulence of pathogens • more linked to breeders – other users

• To communicate more –> Plant Pathologist Network • To exchange the protocols/tools for genotyping • To harmonize the protocols towards a common protocol • To write a manual?

• To make capacity building on these common protocols

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ANNEX 7: PHENOTYPING PATHOGENS

Participants: B. Adamou, N. Kouassi, E. Traoré, T. Kumashiro, Y. Fukuta, N. Vera Cruz, A. Ghesquière, L. Albar What for?

• to facilitate the development of strategies for disease resistance breeding, for specific environments and to identify effective resistance genes against the existing pathogen population;

• to know/understand the diversity of the pathogen population in terms of pathogenicity and to identify tester strain; more importantly, the virulence spectrum of the pathogen population will be understood as well, based on the existing differential varieties carrying specific resistance gene;

• to provide common standarts (differential varieties, methods) for comparative analysis of pathogen population across countries or regions – to harmonize the common methods for pathogen diversity analysis.

How? Pathogen Methods

Viruses

- NIL available for Tungro resistance evaluation; differential accessions (and soon NIL) available for RYMV;

- small differences between the inoculation protocols used in the different labs (for RYMV at least)

Bacteria

- NILs and pyramided lines available for BLB in japonica (partially) and indica – none yet for BLS

- standardized inoculation methods available for sharing for BB and BLS (can be used for high throughput evaluation)

Fungi

- Blast : NILs in indica (Co39, US2) and japonica (LTH) background available

- Standard inoculation methods and assessments are available for blast and brown spot

Nematodes

- protocols exist but need to be simplified into a standard evaluation system

- differential varieties available  

 How to improve?

• need for standard evaluation methods for all the pathogens / when not available • differential lines in same genetic background for all the pathosystems and all the

different genes, individually or in combination • establish a catalogue of the isolines/varieties available

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• collectively decide what are still needed and which institution can take in charge the dvlpt of additional material

• identify a universally susceptible accessions as control (Co39?) and as an appropriate genetic background for NIL development. A mega-variety (very large adaptation for rice growing conditions) as genetic background may be used as research tool but also as pre-breeding material)

• need a focal point for seed distribution • core set of pathogen isolates need to be distributed, when possible

What’s next?

• Short term Functional network of partners need to be established

• Long term: Long term observation of plant/ environment interaction

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ANNEX 8: RICE PATHOLOGY NETWORK  

 

Objectives of the network: ! Optimize research on rice pathology: avoid redundancy and complement each

other ! Identify and fill knowledge gaps ! Create baseline information ! Synthesize information for breeders and different stakeholders ! Create a scientific community ! Detect an emerging disease ! Optimize the search for control strategies  

 

 

Network activities and organization: General network coordinators: D. Silué (AfricaRice, J. Bigirimana (IRRI) & I. Wonni (INERA) Task 1 : Communication - Establishment of a list of people interested in the Network (see Annex 1) - Creation of a dedicated website

Webmaster from IRRI? Coordination team: C. Tollenaere (IRD), R. Oliva (IRRI), E. Traoré (INERA), M. Rakotomala (FOFIFA), M. Sester (CIRAD), ? (IRRI ESA), ? (Africa Rice Center)    

Proposal for the Website architecture - Mission statements - Objectives - Ongoing research projects - List of institutions & members (name, expertise, location) - Rice diseases: description (pictures, distribution), protocols, publications, disease outbreaks - Training - Available facilities and resources - Funding opportunities, ongoing calls of proposal - Links (existing networks, related projects, related websites)

Task 2: Search for funding for network operating costs or associated activities - Prospects on research proposals associating the network Coord.: B. Szurek (IRD) & D. Silué (AfricaRice)

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- Search for funding opportunities GRISP (resp. J. Bigirimana) Agropolis (resp. A. Ghesquière) BMGF (resp. N. Vera Cruz) EU (resp. D. Tharreau) CORAF (resp. T. Kumashiro) AUF (resp. H. Baimey), McKnight (resp. N. Vera Cruz), ABD (resp. N. Kouassi), others : IRD/CIRAD/...  

Task 3: Establishment of standard protocols Protocols for pathogen isolation, conservation, inoculation, phenotyping, genotyping, race designation. General coordinator: D. Silué (AfricaRice) Coordinators by pathogen: Fungi (D. Tharreau, B. Zhou) Bacteria (N. Vera Cruz) Viruses (A. Galzi, M. Rakotomalala) Nematodes (S. Bellafiore) Task 4: Training Coord.: J. Bigirimana (IRRI), D. Silué (AfricaRice) & E. Traoré (INERA) Identification of the needs, identification of funding opportunities, communication on trainings Task 5: Network expansion Coord.: V. Verdier (IRD) Task 6: Research activities Cf: conclusions of the round tables