Microbial Genomic Sequencing Perspectives of the American Phytopathological Society

The American Phytopathological Society 3340 Pilot Knob Road St. Paul, MN 55121-2097 Phone: +1.651.454.7250 Fax: +1.651.454.0766 Email: aps@scisoc.org APSnet: www.apsnet.org

Microbial Genomic Sequencing Perspectives of the American Phytopathological Society

Background Microorganisms play a critical role in plant health. Depending on the organism, they can cause multiple diseases or prevent them. Yet, on a genomic level, we know almost nothing about them (The Microbe Project, 2001,National Science and Technology Council, Office of Science and Technology Policy, Washington, D.C., 29 pg.). To intervene in disease and understand the basis of biocontrol, genomics is as essential for microorganisms in agriculture as is the sequencing of the human genome and Arabidopsis for human health and plant health, respectively. The APS has consulted its members and constituencies on priority setting of microorganisms that should be sequenced. The list below, compiled in 2000-2001, is subject to change. Nevertheless, there is a critical void in this area. Plants for food, fiber, forestry and quality of life are subject to losses caused by a variety of physical and biological factors. Biological agents that cause losses estimated at 2035%, at over $200 billion annually, are insects and microbes. Due to multiple factors, such losses will continue to be a challenge. These factors include global climate change, plants grown in new ecosystems, mutation or gene transfer in pathogens, and spread of pathogens by nature or by commerce (shipping, transport, and storage). Such losses are considered unsustainable in view of projected needs of human and animal populations. Traditional plant breeding programs and projected programs based on newer genetic approaches have made and will make a significant impact on reducing losses; however, plant diseases remain a critical and continuing threat and biocontrol with microorganisms still a small segment of management. Genomics, or the ability to analyze whole genomes, offers unprecedented opportunities to obtain new information that will allow deployment of effective disease management programs with enhanced safeguards to the environment. A complete understanding of disease susceptibility and resistance will require understanding the interactions between plant hosts and pathogen or biocontrol genotypes. Sequence data from the genes for such microorganisms is essential to advance our knowledge of infection and the interaction of pathogens and host gene products, and will provide knowledge on how these organisms reproduce and spread. Effective control will then be achieved through developing host resistance or through means that combat the pathogen directly or indirectly. The use of genomics has already been demonstrated through enhancing disease resistance by using viral components that can be incorporated into a host with current technology. Identifying critical genes in other pathogens will permit similar strategies to be developed for disease control. Similar strategies are receiving significant support for microbes of medical importance, as such efforts are identifying targets for drugs, and suggesting new targets for drug development and diagnostics. The genetic potential and sensitivity of pathogens must be understood to effectively combat current and future disease threats.

Priority Setting and Criteria A variety of organisms cause and prevent plant diseases. Fungi and viruses are the most significant and widespread. It is estimated that there are more than 10,000 pathogenic species of fungi and viruses, many of which are economically important. It is clear that it is not possible to obtain genomic sequences of all of them, at least based on current and anticipated technology. Thus, we suggest focusing on a representative set of significant microorganisms. For genome sequences to be of optimal value for functional studies, it is estimated, from comparative studies of bacterial genomes, that the genomes of at least 10 related organisms are required. In broad categories, we make such recommendations and have compiled a list of such organisms. The listing has been developed using these criteria, essentially as outlined by the USDA (RFP for IFAFS): Economic importance and relevance to U.S. (and world-wide) agriculture. Unique biological or environmental features. Broad interest to a significantly sized community of scientists or agriculturists. Genetic tractability, i.e. the ease with which genetic studies, such as crosses, genome modifications, etc. can be performed. Availability of tools and other biological resources, such as gene libraries, genetic maps, genetic tractability of the host so that the system could be addressed from both sides of the host-pathogen interaction,etc. It is recognized that some organisms may be of critical agricultural importance but not easily cultured or subjected to genetic analysis, and therefore may be candidates for sequencing. Needs Additional and new funding is of critical need. A growing number of research programs are at a major impasse due to lack of information about pathogen and biocontrol genomes. Cloning and characterization of individual genes is no longer cost effective. Microorganisms in general have compact genomes, in most cases a fraction of the size of plants and animals. Costs of genomic sequencing are still high, but decreasing. Functional and structural genomics will be the next phase of analysis. Some investment has occurred in the private sector, but relatively little. Very little has been publicly available for plant pathogens. The lack of sequence information for agriculturally important microorganisms in public databases has other serious consequences, such as those related to human resources,trade (phytosanitary) issues, and agroterrorism. To date, the complete sequences of many plant viruses, and one bacterium, Xylella fastidiosa are known.

Compiled List of Prioritized Fungal and Oomycete Species for Genomic Sequencing Priority Criteria* Priority 1 Species Magnaporthe grisea Common Name rice blast fungus Genome Size 40 Mb Rationale or Significance a) Most devastating fungal disease of rice b) Amendable to classical and molecular genetics a) Most important pathogen of potato b) Model system within the oomycetes c) Advanced genetics and transformation system and libraries a) Causes one of the most important plant diseases in US, wheat head scab. b) Produces trichothecene mycotoxins. c) Studied by large number of investigators worldwide. Important producer of aflatoxins, extremely potent carcinogenic mycotoxin. Important basidiomycete model system for the smuts which are economically important disease agents. a) Causes rust disease on several agriculturally important cereal crops (barley, oat, rye and wheat). b) Distributed worldwide

Phytophthora infestans

potato late blight fungus, cause of the Irish potato famine wheat head scab fungus

237 Mb

Fusarium graminearum

40 Mb

4 5

Aspergillus flavus Ustilago maydis corn smut fungus

~ 30 Mb 20 Mb

Puccinia graminis

cereal rust

67 Mb

Compiled List of Prioritized Fungal and Oomycete Species for Genomic Sequencing Priority Criteria, Continued Priority 7 Species Fusarium moniliforme (verticillioides) Cochliobolus heterostrophus Botrytis cinerea Common Name Genome Size 46 Mb Rationale or Significance a) Major pathogen of maize worldwide, causes both kernel and stalk rot. Gibberella teleomorph b) Produces fumonisin mycotoxins Important model and historically significant desease. Economically very significant pathogen and taxonomically distinct discomycete Botryotinia teleomorph Glomerella teleomorph

8 8

southern corn leaf blight gray mold

10 11 12 12

Colletotrichum graminicola Mycosphaerella graminicola Cryphonectria parasitica Epichloe sp.

chestnut blight fungus grass endophyte

29-35 Mb

a) Endophytes provide benefits to forage grasses but often reduce livestock productivity. b) The model endophyte, E. festucae is the most genetically tractable, plant-mutualistic fungus and has numerous agronomically important genes such as for biprotective alkaloids.

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Pythium ultimum

Compiled List of Prioritized Fungal and Oomycete Species for Genomic Sequencing Priority Criteria, Continued 15 Sclerotinia sclerotiorum white mold a) Number 2 cause of yield loss in soybean in US b) Major pathogen of canola, sunflower, edible beans, vegetable crops and more than 400 plant species. a) Has a wide geographic distribution. b) Is an economically important pathogen of many plant species, including maize, potato, rice, soybean and wheat. Important symbiotic fungus

15

Rhizoctonia solani

37-46 Mb

17 18 19 20 21 21 23 24 25 26

Blumeria graminis Penicillium Glomus sp. Venturia inaequalis Tilletia caries Heterobasidium annosum Cercospora kikuchii Armillaria mellea Aphanomyces euteiches Cronartium ribicola

powdery mildew mycorrhizal fungus apple scab Common bunt/ stinking smut 100 Mb

28.4 Mb

a) Significant foliar and seed pathogen of soybean b) World-wide in occurrence.

* The list provided was generated with extensive input from the following APS committees; Biochemistry, Physiology, and Molecular Biology; Genetics; Mycology; and Mycotoxicology. Each committee independently ranked the 26 species originally provided by the four committees to the APS Public Policy Board. Rankings from individual committees were compiled and a new unified ranking generated.

The rankings were quite consistent between the committees indicating that there was a general consensus regarding the relative importance of species. This was especially true of the top 5 species. Species ranked 6-9 clearly clustered with each species more or less equal in perceived merit. Finally, it was recognized that there are numerous fungi and oomycetes that merit consideration for genomic sequencing efforts and it is also well understood that priorities must be placed on some species. It is critical to note that circumstances beyond the current knowledge of the committees may, at a future date, significantly increase or decrease the importance of genomic sequencing efforts of particular species listed and also that species not included may come to the forefront of importance. Another issue not directly addressed in these rankings is taxonomic placement of species and this could also influence priorities in the future. Thus this list is a best effort to rank fungi and oomycetes by relative importance based on the criteria. This list should not, however, be viewed as static or permanent. This list should be updated with further broad input periodically.

Compiled List of Priority Bacteria for Sequencing* Organism (by category) E. amylovora Common Name fire blight Genome Size Ca. 4.5 Mb

Strain Ea273

Streptomyces scabies Pantoea stewartii subsp. stewartii

Potato strain Wild type

wart/russet on potato wilt on corn

Not known Ca. 4.7 Mb

Rationale or Significance a) Most imporant bacterial disease of apples, pears, and related perennials worldwide. b) A model pathosystem for understanding host: parasite interactions, ecology of plant pathogens, and parasitic fitness. c) Proposal in now to USDA/NSF a) An actinomycete pathogen of major economic concern in potato production; b) Quarantine problem a) Serious pathogen of corn and is of high interest because it is vectored by an insect species. b) High priority strain for genome scale comparison with enterobacterial pathogens to elucidate putative virulence factors and compare the evolutionary history of pathogenesis.

Compiled List of Priority Bacteria for Sequencing, Continued Erwinia carotovora subsp. Carotovora (Pectobacterium carotovorum subsp. Carotovorum) Wild type soft rot disease Ca. 4.0 Mb a) Serious pathogen causing post-harvest rot of important food crops. b) Few effective control strategies available and detection technologies are foundational to management. c) High priority strain for genome scale comparison with enterobacterial pathogens to elucidate putative virulence factors and compare the evolutionary history of pathogenesis.

* The list provided was generated by the Bacteriology Committee. All four species were considered of high priority by the committee.

Compiled List of Priotized Mollicutes for Genomic Sequencing Priority 1 2 3 4 5 Species Spiroplasma kunkelii Western X phtyoplasma Spiroplasma citri Aster yellows phytoplasma S. floricola Strain CR-2 Wild type BR33X Wild type 23-6 Common Name corn stunt spiroplasma none none none none Genome Size 2400 kb 670 kb 1600 kb 1100 kb 2600 kb Rationale or Significance Major pathogen of corn; genome is now being sequenced and is almost complete. Taxonomic group I. Major pathogen of stone fruits; currently being sequenced in the U.S.; different taxonomic group than aster yellows phytoplasma; genome is one of the smallest of all mollicutes. Major pathogen of citrus and brassicaceous plants; used as model system for biology, pathogenicity and insect transmissibility of phytopathogenic mollicutes. Taxonomic group I. Major pathogen, large plant host range; currently being sequenced in the U.S.; part of a large taxonomic group. Important for comparison since this species is an epiphyte but not a pathogen; also in a different taxonomic group than the other two spiroplasmas (group III). 8

Compiled List of Priority Viral Genera for Sequencing*

Organism (by category) 1 Potyvirus/

Strain MDMV BCMV SMV

Common Name Maize dwarf mosaic Bean common mosaic Soybean mosaic Potato Y Wheat streak mosaic Citrus tristeza, Tomato chlorosis,

Genome Size 10.4 kb

Rationale or Significance Largest single taxonomic group of plant viruses, transmission by aphids (nonpersistent manner), whitefly or fungi, some seedborne, include leguminous, solanaceous and monocot hosts, global distribution.

Tritimovirus 2 Closteroviruses/ Criniviruses 3 Begomoviruses/

PVY WSMV CTV ToCV PMWV

17-20 kb/15kb

Curtoviruses 4 Tospoviruses 5 Tombusvirus

Pineapple mealy bug wilt SLCV, Diverse strains and BGMV, species: Bipartite AbMV, New World or Old PHV World monopartite clades viruses of cultivated BCTV and and wild species related viruses TSWV Tomato spotted wilt TBSV CNV Tomato bushy stunt Cucumber necrosis

Viruses of woody and herbaceous hosts, large complex genomes of ssRNA, semipersistent, associated with hemipteran vectors (aphid, mealy bug and whitefly). Only circular ssDNA plant viruses, global, greater than expected variability, apparent rapid evolution through recombination and reassortment, poor association between viruses of cultivated and weed hosts, several hemipteran vectors

5.2 kb

2.8 kb ~17 kb 4.8 Large, complex, ssRNA virus replicates in insect vector. Medical significance (Bunyaviridae). Highly stable, ssRNA viruses transmitted in persistent manner by fungal zoospores

Compiled List of Priority Viral Genera for Sequencing*

Organism (by category) 6 Luteovirus/ Polerovirus 7 Cucumovirus 8 Rhabdovirus

Strain BYDV BWYV CMV strains SYVV

Common Name Barley yellow dwarf Beet western yellows Cucumber mosaic Sonchus yellow vein

Genome Size 5.5 kb

Rationale or Significance SsRNA viruses transmitted in persistent, circulative manner by aphids, worldwide distribution, infect monocots and dicots.

8.0 kb 13.0 kb

One of the largest groups of plant viruses, have broad host ranges, but poorly understood biodiversity. Global distribution. Large, complex, membrane bound RNA viruses, replicates in insect, medical significance through relationship to animal viruses.

* The Virology Committee has expressed strong interest in including plant viruses in the microbial genome effort. Plant viruses offer a unique opportunity to study pathogen genome sequence and function and to relate this to genomic studies with host plants. Plant viruses, more than any other plant pathogen, maintain an intimate relationship with their host functions. While the Virology Committee fully recognized the importance of plant virus diseases as serious threats to plant health and associated economic losses, it wishes to stress the importance of knowledge about gene function and the uniquely interesting interaction between the virus and its host. Such studies not only serve to dissect the biological characteristics of the virus, but are used to understand biological/physiological functions of the host. An excellenct example is the process of cell-to-cell movement by viruses where a viral protein(s) interacts with host cellular connections, plasmodesmata, allowing virus to move from one cell to another. These studies allow better understanding of the physiological processes associated with plasmodesmata and the trafficking of molecules from one cell to another. Furthermore, because a virus' genome is relatively simple, the processes of obtaining genomic sequence and functional genomic data will be more rapid and straightforward than other microbial pathogens.

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Compiled List of Priority Nematode for Sequencing* Priority 1 2 3 4 5 6 7 8 9 10 Organism (by category) Meloidogyne incognita Pratylenchus penetrans Globodera pallida Ditylenchus dipsaci Meloidogyne hapla Heterodera glycines Radopholus similus Aphelenchoides fragariae Nacobus abbesans Rotylenchulus rentiformis Common Name Southern rootknot nematode Lesion nematode Potato cyst nematode Stem nematode Northern root knot nematode Soybean cyst nematode Burrowing nematode Foliar nematode False root knot nematode Reniform nematode Genome Size 5.1 x 107 1 X 108 Rationale or Significance Most economically important nematode worldwide. A sedentary endoparasite that has an extremely wide host range. An economically important migratory endoparasite. Wide host range with a different feeding habit. Most economically important nematode in Europe. Sedentary endoparasite of potato. One of the most devastating nematodes on a wide range of crops worldwide. Feeds on the shoots of plants as a migratory endoparasite. Problems on wide range of crops such as alfalfa, vegetable; cool weather nematode. Has diploid strains. Most economically important pathogen on soybean. Chosen as a model nematode for plant parasites. Sedentary endoparasite. An economically important migratory endoparasite on citrus and ornamentals in the Americas. A leaf-inhabiting nematode that is a pathogen on ornamentals. Problems in Western US on potato, same family as Pratylenchus and Radopholus but sedentary. Widely distributed sedentary semiendoparasitic nematode in the tropical and subtropical countries that is an important parasite with a wide host range.

9.2 x 107 -

* Compiled by the nematode committee.

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