Molecular Analysis of the Wheat Blast Population in Brazil with a Homolog of Retrotransposon MGR583

日植病報 65: 429-436 (1999) Ann. Phytopathol. Soc. Jpn. 65: 429-436 (1999)

Alfredo S. URASHIMA*,**,Yoko HASHIMOTO*,Le D. DON*,Motoaki KUSABA*,***, Yukio TOSA*,Hitoshi NAKAYASHIKI*and Shigeyuki MAYAMA*

Abstract Wehave identified a familyof dispersedrepetitive DNA sequences in the genomeof a Magnaporthe griseaisolate from finger millet (Eleusine coracana). Southern blot analysesshowed that thiselement is presentin a moderatecopy number (30-40 copies) in thegenome of blastisolates from wheat (Triticum aestivum).DNA sequence data suggestedthat this elementcontains a regionhighly homologous to the reversetranscriptase domain of MGR583,a poly A-type retrotransposon. Using the reversetranscriptase domainof this elementas a molecularprobe, the geneticstructure of the wheatblast population was examined.DNA fingerprinting analyses revealed that the wheat-infectingisolates constitute a separate, singlelineage of their own,suggesting that theyare derivedfrom a singleorigin. (ReceivedOctober 26, 1998; Accepted April 23, 1999) Key words: Magnaporthegrisea, blast fungus, retrotransposon, wheat, DNA fingerprinting.

that the wheat pathogen is distinct from the rice patho- INTRODUCTION gen. This difference between the rice pathogen and the wheat pathogen has raised questions regarding the Magnaporthe grisea (anamorph, Pyricularia grisea) is a origin of the wheat pathogen and its relationship to common pathogen of monocots that infects more than isolates from other hosts. Based on the pathogenic 50 grass species18,29). Among them, rice (Oryza sativa) is pattern on a set of gramineous plants, Urashima et al.38) the most important staple crop for which this fungus have noted that blast isolates from finger millet causes major losses, with frequent reports of devastat- (Eleusine coracana) are the most similar to the wheat ing damage worldwide. Recently, another crop of vital pathogen. Restriction fragment length polymorphism importance has been reported to be suffering from high detected by single-copy probes has also shown a close yield loss because of this fungus in Brazil. In 1985 some relationship between isolates from finger millet and wheat (Triticum aestivum) fields in Northern Parana those from Brazilian wheat when compared with those state were severely damaged by this pathogen13). Since from corn or rice21). These results were curious because this first report, the wheat blast disease has spread to finger millet is an unknown crop in Brazil. other regions4,9,12,30,31),and it is now present in all major MGR58611) is the most frequently employed probe in wheat-growing regions of this country. In the state of molecular studies of the blast fungus. It belongs to the Mato Grosso do Sul the yield reduction has been esti- inverted repeat (IR) class of transposon5) and is present mated to be 10 to 11%7). It is currently considered to be in a high copy number in rice isolates but in a low copy one of the major wheat diseases in Brazil as a result of number in isolates from wheat and weeds. Using this the damage it inflicts, its geographical distribution, the probe, Valent40) has demonstrated the difference poor performance of chemicals in protecting wheat between the rice pathogen and the wheat pathogen. spikes in the field and the lack of resistant cultivars. Further, MGR586 has proved to be a reliable genetic Because this disease occurred in a region where wheat marker for studies of epidemiology and population and rice are cultivated in close proximity, wheat blast structure of the rice blast pathogen in various parts of was soon suggested to be caused by the rice blast the world, allowing the identification of clonal pathogen13). Later reports, however, have provided infor- lineages1,22,23,32,35,42,44).However, its usefulness in study- mation on differences between these pathogens. Studies ing the genetic aspect of the wheat pathogen is limited on the host range31,38)and sexual fertility38) have shown by its low copy number in the wheat pathogen

* Faculty of Agriculture , Kobe University, Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan 神戸大学農学部 ** Present address: Department of Plant Biotechnology , Center of Agricultural Sciences, Federal University of Sao Carlos, P.O. Box 153, 13600-000 Araras, S.P., Brazil *** Present address: Faculty of Agriculture , Saga University, Honjo 1, Saga 840-8502, Japan 現在:佐賀大学農学部 430 日本植物病理学会報第65巻第4号平成11年8月 genome10,40). Besides MGR586, many repetitive DNA sequences MATERIALS AND METHODS have been found in the genome of M. grisea2,6,11,15,16,33,36). Fosbury33) and MAGGY6), retrotransposons containing Collection, identification and culture conditions LTRs, have common characteristics of high copy num- of isolates A total of 77 isolates of M. grisea were ber in isolates from rice and foxtail millet, but being collected from wheat [Triticum aestivum (L.) Thell.] absent in those from other hosts37), which make them and weeds in Parana (PR), Sao Paulo (SP) and Mato adequate markers for studies of the rice blast Grosso do Sul (MS), Brazil, where the wheat blast population3,19) but not for the wheat blast population. disease is prevalent. The following diseased grass weeds Grasshopper, another retroelement present in multiple were gathered: Digitaria horizontalis Willd., Brachiaria copies, is dispersed throughout the genome of some plantaginea (Link) Hitchc., Cenchrus echinatus L., isolates from Eleusine spp. but is absent in isolates from Setaria geniculata (Lam.) Beauv. and Echinochloa colo- other hosts2). MGR58311) is present in a high copy num- num Link. Isolates from rice [Oryza sativa L.] collected ber in rice isolates and in a majority of non-rice in Brazil or Japan and those from finger millet [Eleusine isolates14) but shows a less polymorphic hybridization coracana (L.) Gaertn.] collected in Japan, Nepal or India pattern compared with that of MGR58641). were also tested. The designation, original hosts, geo- The present work provides a molecular approach to graphic origin and date of collection of the isolates are the study of the wheat blast population. For this pur- shown in Table 1. Three samples were taken from each pose, we isolated and characterized a molecular probe to wheat field, which was surrounded by rice fields or had use to examine the population structure of the wheat rice plants grown in the same field. All isolates were pathogen and its genetic relationship to isolates from confirmed to be pathogenic to its own host. In the test of other hosts. pathogenicity of wheat isolates, Triticum aestivum cv.

Table 1. Host and locality of Magnaporthe grisea isolates tested

a) Isolates with the same number followed by different alphabetical letters were collected from the same field. Ann. Phytopathol. Soc. Jpn. 65 (4). August, 1999 431

Anahuac was used as a test plant. Monoconidial cultures of these isolates were grown on sterilized barley seeds in a vial, then dried throughly at 25•Ž and kept in a case with silica gel at 5•Ž. DNA manipulation Isolates were grown in CM broth (3g Casamino acids, 3g Yeast extract, 5g su- crose/l) on a rotary shaker at 25•Ž. After 4-5 days incubation, mycelia were harvested and stored at -80•Ž. DNA was extracted following the procedure described by Liu et al.24) with a few adaptations. A repetitive DNA sequence for the probe was isolated from genomic DNA of an isolate from finger millet,

G10-1. It was inserted into the plasmid pUC119 and established as pEBA18 in E. coli strain C75 (Takara Shuzo Co.). Its 0.9-kb fragment was subcloned into pUC119, established as pEBA18-09 and subsequently sequenced using the Auto Read Sequencing Kit (Phar- macia Biotech.), according to manufacturer's instruc- tion. The subclone was labeled with biotin using NEBlot Phototope Kit (New England Biolabs) to use as a probe for fingerprinting. One microgram of genomic DNA was digested to Fig. 1. DNA fingerprints of Magnaporthe grisea isolates completion with EcoRI and separated by horizontal from various hosts. DNA was digested with agarose gel (0.8%) electrophoresis at 2V per centimeter EcoRI and hybridized with pEBA18-09. The in a 0.5X TBE buffer for 18hr. DNA fragments were lanes contain genomic DNA from: 1, Br37; 2, denatured and transferred to a nylon membrane (Magna- Br38; 3, Br36; 4, Br31; 5, Br30; 6, Br27; 7, Graph, Micron Separations, Inc.) by overnight capillary IN77-31-1-1; 8, NP10-17-4-1-3; 9, Z2-1; 10, Bp3a; blotting. Prehybridization and hybridization were con- 11, BR35; 12, Br34; 13, Br116.5; 14, Br48; 15, Br7; 16, Br13; 17, Br10; 18, 0903.4. Host plants ducted in 6X SSC containing 5X Denhardt's solution, of these isolates are indicated below the panel: 0.5% SDS and 100ƒÊg/ml of sonicated salmon sperm Sg, Setaria geniculata; Eh, Echinochola colo- DNA in a sealed plastic bag at 68•Ž. Hybridization num; Ce, Cenchrus echinatus; Dh, Digitaria reactions were allowed to continue for 12-15hr with the horizontalis; Ec, Eleusine corecane (finger biotin-labeled probe. After hybridization, the membrane miller); Bp, Brachiaria plantaginea; Ta, Triti- was washed twice in 2X SSC, 0.1% SDS for 5min each cum aestivum (wheat); Os, Oryza sativa (rice). time at room temperature, followed by two washes in Kilobase markers (kb) are indicated on the right 0.1X SSC, 0.1% SDS for 15min at 68•Ž. DNA blots were side. Arrowheads represent common bands submitted to chemiluminescent detection using Photo- shared by wheat isolates and Brachiaria iso- topeTM Star Detection Kit (New England Biolabs). lates. Analysis of fingerprints The band pattern of DNA fingerprints was scored visually by checking the presence (value 1) or absence (value 0) of bands in the 1- 7kb range. Bands with ambiguous interpretation were RESULTS not evaluated in order to minimize unexpected errors. Similarity coefficients were calculated by Nei and Li's Isolation and partial characterization of method27): Sxy=2Nxy/(Nx+Ny), where Nxy=number of pEBA18 fragments shared by two isolates, and Nx and Ny= Genomic DNA of a finger millet isolate, G10-1, was number of fragments in isolate x and y. A phenogram digested with HindIII and cloned into pUC119. One of was constructed using the •gunweighted pair group these clones, pEBA18, proved to be present in multiple method using arithmetic means•h (UPGMA)34) with a copies in wheat isolates (Fig. 1). The insert of pEBA18 computer program developed by M. Okuda, Kyushu was a 5.5-kb HindIII fragment (Fig. 2). A 0.9-kb EcoRV- National Agricultural Experiment Station, Japan. A HindIII fragment of the insert was then subcloned into bootstrap analysis was carried out with the WINBOOT pUC119 and established as pEBA18-09 for sequencing. program43), and the robustness of each cluster was Its complete sequence and deduced peptide sequence are verified in 1000 replications. shown in Fig. 3. pEBA18-09 showed a very high homol- ogy (98.6% in nucleotide sequence) to the reverse transcriptase domain of a non-LTR retrotransposon, MGR583, reported by Hamer et al.11), and Valent and Chumley41) (Figs. 2 and 3). 432 日本植物病理学会報第65巻第4号平成11年8月

Fig. 2. Maps of MGR583 and pEBA18. The hatched box represents a region of MGR583 corresponding to pEBA18-09, a subclone of pEBA18. H, HindIII; Sm, SmaI; E, EcoRV; B, BamHI; P, PstI; C, ClaI; X, XhoI; S, SalI.

Fingerprinting with the MGR583 homolog Genomic DNA of blast isolates from various hosts DISCUSSION was digested with EcoRI, electrophoresed, and hybridized with pEBA18-09. Isolates from rice carried this The main objectives of this work were to isolate a element in a high copy number (>40 copies), whereas clone that allows DNA fingerprinting of non-rice isolates those from wheat, finger millet and Digitaria, etc. car- of M. grisea and to examine the genetic variation in M. ried it in a moderate copy number (20-40-copies) (Fig. 1). grisea populations that infect wheat and weeds in Brazil. An isolate from Setaria geniculata carried only a single Analyses of the population structure of the blast fungus copy (Fig. 1). When hybridization profiles were ex- using DNA fingerprinting have been conducted in many amined in detail, isolates from Digitaria horizontalis, parts of the world1,3,22,23,32,35,42,44).Those studies have Echinochloa colonum, Cenchrus echinatus and finger focused exclusively on the rice pathogen, and most of millet appeared to be distinct from wheat isolates them have used MGR586 as a molecular probe. This because they produced no common bands with wheat probe and others described so far have not been suitable isolates. Isolates from Brachiaria plantaginea produced for examining genetic variation in the wheat blast four common bands with wheat isolates. To clarify the population. In the present study we isolated a repetitive relationship between Brachiaria isolates and wheat iso- element from a finger millet isolate as a molecular lates, a phenogram was constructed using UPGMA (Fig. marker for the wheat blast population. A 0.9-kb frag- 4). Each population formed a robust cluster with a high ment of this element has proven to be a homolog of the bootstrap value, and the similarity coefficient between reverse transcriptase domain of MGR583, a non-LTR them was only 16%, suggesting that each represents a retrotransposon found in rice isolates by Hamer et al.11). distinct genetic group. However, confidence of clusters It was present in a moderate number (30-40) in the within the wheat blast population was low, indicating genome of wheat isolates, and its use as a molecular that the wheat blast population has a high variation and probe allowed us to examine their genetic variability. cannot be separated into lineages. DNA fingerprinting with this probe revealed that DNA fingerprints with pEBA18-09 were further ana- wheat isolates collected in different regions of Brazil lyzed to elucidate the population structure of wheat were clustered into a single group; all the isolates tested isolates collected in major regions of Brazil where blast shared more than 75% of the bands with one another, disease was prevalent. All these isolates shared more but no or few bands with isolates from weeds or finger than 75% of the bands (Figs. 1 and 4). However, patterns millet. In other words, the wheat isolates constituted a of uncommon bands showed a high degree of diversity. separate, single lineage of their own. These results Diverse haplotypes occurred even in a population col- suggest that the wheat blast population in Brazil is lected from the same city and year. For example, iso- derived from a single origin. In the subsequent dissemi- lates Br130.1B, Br130.1D, Br130.1E, Br130.8, Br130.9B nation to new regions, seed transmission seems to have and Br130.90 collected in Rolandia (PR) in 1992, formed played an important role because wheat seeds have a group with only 81% similarity. Such diversity was frequently been found to be contaminated with the blast more evident when isolates from the same field showed fungus8,20,25,26). a similarity as low as 78% (isolates Br119.1B, Br119.1C, Nevertheless, the wheat blast population showed a Br119.1F). On the other hand, 100% similarity occurred high degree of variation among isolates. Unique (uncom- among isolates collected in different locations of the mon) bands appeared in various combinations. Conse- same state (Br48 from Itapora, MS, and Br50 from Rio quently, no lineages were detected within the wheat Brilhante, MS), those gathered in the same state but in blast population in contrast to the structure of the rice different years (Br2 and Br3 from Londrina, PR, sam- blast population that is composed of distinct clonal pled in 1990, and Br130.1E from Rolandia, PR, collected lineages1,3,22,23,32,35,42,44).We can point out two factors in 1992) and those collected in the same year, but in which may be related to this difference between the rice different states (Br202.1D from MS and Br123.1D, Br- blast and wheat blast populations. One is the history of 127.11C, Br127.11D, and Br128.1B from PR). the blast diseases. The rice blast pathogen, with its long Ann. Phytopathol. Soc. Jpn. 65 (4). August, 1999 433

Fig. 3. Comparison of nucleotide sequences and putative amino acid sequences between pEBA18-09 (abbreviated as pEBA18, accession # AB025252) and a corresponding region of MGR583 (MGL, accession # AF018033). 434 日本植物病理学会報第65巻第4号平成11年8月

An isolate from S. geniculata produced only one hybridization band with pEBA18-09, suggesting that it is remote from wheat isolates. Although this isolate has been shown to be pathogenically similar to the wheat blast pathogen with positive cross-infection39), there was no sexual compatibility between them38). Isolates from D. horizontalis, C. echinatus and E. colonum produced no common bands with wheat isolates (Fig. 1), suggesting that those isolates are not close to wheat isolates. This result is in accordance with pathogenicity data39) that isolates from those weeds are incompatible with wheat and vice versa. Finger millet isolates also had no bands in common with wheat isolates, suggesting that they are not so closely related to wheat isolates. This result was unexpected because the former work38) has verified a close resemblance between wheat isolates and finger millet isolates in terms of host range and sexual compatibility. However, the present result appears to be logical, since finger millet, which is a staple food in Africa, India, Nepal and other regions of Asia, is absent in Brazil. Brachiaria plantaginea isolates shared four bands with wheat isolates but produced a cluster with only 16% similarity to the wheat blast population, suggesting that they form a distinct group. The usefulness of pEBA18-09 for studying the population structure of the wheat blast pathogen has been demonstrated in the present work. This probe also appeared to be applicable to fingerprinting of various weed isolates. We suggest that the MGR583 homolog, and probably MGR583 itself, promise to be useful probes for fingerprinting M. grisea isolates from a broad range of hosts and to be good complements to the MGR586 element which is somewhat limited in its usefulness.

Fig. 4. UPGMA dendrogram of Brachiaria isolates We thank Dr. R. Nelson, the International Rice Research (Br34, Br54, Bp3a, and Br35) and wheat isolates Institute (IRRI), for providing the WINBOOT program. The (others) collected in Brazil based on fingerprints first author, a postdoctoral fellow, is indebted to the Japan with pEBA18-09. Values at cluster branches International Cooperation Agency (JICA) for financial sup- indicate results of the bootstrap analysis. port. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (Nos. history of interaction with rice plants and agricultural 10556011 and 10460022). practices, has suffered from selection pressures exerted by cultivar resistance, chemical application and so on. In Literature cited contrast, the history of the wheat blast is very short; its first report was as recent as 1986. The other factor is 1. Chen, D., Zeigler, R.S., Leung, H. and Nelson, R.J. (1995). mating capacity. The sexual stage of M. grisea seems to Population structure of Pyricularia grisea at two screen- have no effect on the population structure of the rice ing sites in the Philippines. Phytopathology 85: 1011- blast pathogen because of its low sexual fertility17,41). By 1020. 2. Dobinson, K.F., Harris, R.E. and Hamer, J.E. (1993). contrast, the wheat blast pathogen has a much higher Grasshopper, a long terminal repeat (LTR) retroelement fertility, which is expressed by high sexual compatibility in the phytopathogenic fungus Magnaporthe grisea. Mol. with isolates from various hosts as well as within wheat Plant-Microbe Interact. 6: 114-126. isolates38). Moreover, compatible mating types have 3. Don, L.D., Kusaba, M., Urashima, A.S., Tosa, Y., Naka- been found within wheat isolates from the same yashiki, H. and Mayama, S. (1999). Population struc- location38). The importance of sexual reproduction in the ture of the rice blast fungus in Japan examined by DNA wheat blast pathogen has also been stressed by Orbach fingerprinting. Ann. Phytopathol. Soc. Jpn. 65: 15-24. et al.28). Further observations are needed to determine 4. Dos Anjos, J.R.N., Da Silva, D.B., Charchar, M.J.D. and whether sexual recombination plays a role in the Rodrigues, G.C. (1996). Ocorrencia de brusone diversification of the wheat blast population in Brazil. (Pyricularia grisea) em trigo e centeio na regiao dos Ann. Phytopathol. Soc. Jpn. 65 (4). August, 1999 435

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range, mating type and fertility of Pyricularia grisea 和文摘要 from wheat in Brazil. Plant Dis. 77: 1211-1216. 39. Urashima, A.S. and Kato, H. (1998). Pathogenic rela- Alfredo S. URASHIMA・橋本容子・Le D. DON・草場基章・ tionship between isolates of Pyricularia grisea of wheat 土佐幸雄・中屋敷均・眞山滋志:ブラジルにおけるコムギいもち and other hosts at different host developmental stages. 病菌個体群のレトロトランスポゾンMGR583ホモログによる Fitopatol. Bras. 23: 30-35. 分子解析 40. Valent, B. (1990). Rice blast as a model system for コムギいもち病菌個体群の分子解析のためのプローブとし plant pathology. Phytopathology 80: 33-36. 41. Valent, B. and Chumley, F.G. (1991). Molecular genetic て,シコクビエいもち病菌のゲノムから散在反復配列のファミ analysis of the rice blast fungus Magnaporthe grisea. リーをクローニングした。この因子はコムギいもち病菌にゲノ Annu. Rev. Phytopathol. 29: 443-467. ムあたり30から40コピー存在した。シークエンスの結果,本因 42. Xia, J.Q., Correll, J.C., Lee, F.N., Marchetti, M.A. and 子はポリAタイプレトロトランスポゾンMGR583の逆転写酵 Rhoads, D.D. (1993). DNA fingerprinting to examine 素ドメインと高い相同性を有することが判明した。本因子をプ microgeographic variation in the Magnaporthe grisea ローブとしてコムギいもち病菌個体群のフィンガープリント解 (Pyricularia grisea) population in two rice fields in 析を行ったところ,本菌群はそれ自身で独立かつ単一のリネー Arkansas. Phytopathology 83: 1029-1035. ジを形成していることが明らかとなり,単一起源であることが 43. Yap, I. and Nelson, R.J. (1996) . WINBOOT: a program 示唆された。 for performing bootstrap analysis of binary data to