Origins of host-specific populations of the blast pathogen Magnaporthe oryzae in crop domestication with subsequent expansion of pandemic clones on rice and weeds of rice

Rice, as a widely and intensively cultivated crop, should be a target for parasite host shifts and a source for shifts to co-occurring weeds. Magnaporthe oryzae, of the M. grisea species complex, is the most important fungal pathogen of rice, with a high degree of host specificity. On the basis of 10 loci from six of its seven linkage groups, 37 multilocus haplotypes among 497 isolates of M. oryzae from rice and other grasses were identified. Phylogenetic relationships among isolates from rice (Oryza sativa), millet (Setaria spp.), cutgrass (Leersia hexandra), and torpedo grass (Panicum repens) were predominantly tree like, consistent with a lack of recombination, but from other hosts were reticulate, consistent with recombination. The single origin of rice-infecting M. oryzae followed a host shift from a Setaria millet and was closely followed by additional shifts to weeds of rice, cutgrass, and torpedo grass. Two independent estimators of divergence time indicate that these host shifts predate the Green Revolution and could be associated with rice domestication. The rice-infecting lineage is characterized by high copy number of the transposable element MGR586 (Pot3) and, except in two haplotypes, by a loss of AVR-Co39. Both mating types have been retained in ancestral, well-distributed rice-infecting haplotypes 10 (mainly temperate) and 14 (mainly tropical), but only one mating type was recovered from several derived, geographically restricted haplotypes. There is evidence of a common origin of both ACE1 virulence genotypes in haplotype 14. Host-haplotype association is evidenced by low pathogenicity on hosts associated with other haplotypes.     

Host Species-specific Repetitive DNA Sequence in the Genome of Magnaporthe grisea,the Rice Blast Fungus

We cloned a repetitive sequence to show RFLPs in the genome of Magnaporthe grisea, a fungal pathogen responsible for rice blast. As the sequence was 0.8 kb in length and dispersed in the genome, it was named MGSR1 (for Magnaporthe grisea short repeat 1). MGSR1 was conserved highly in the genome of rice pathogens, but poorly in the genome of pathogens of grasses other than rice. And the RFLPs, displayed with the sequence, could distinguish between clonal lineages in rice-pathogenic isolates. The nucleotide sequence showed the presence of an internal promoter of RNA polymerase III, a 3?-poly(T), and an 8-bp direct repeat in it.     

Population Structure of Magnaporthe grisea from North-western Himalayas and its Implications for Blast Resistance Breeding of Rice

Neutral and pathogenicity markers were used to analyse the population structure of Magnaporthe grisea rice isolates from the north‐western Himalayan region of India. Random amplified polymorphic DNA (RAPD)‐based DNA fingerprinting of 48 rice isolates of M. grisea with five primers (OPA‐04, OPA‐10, OPA‐13, OPJ‐06 and OPJ‐19) showed a total of 65 RAPD bands, of which 54 were polymorphic. Cluster analysis of 48 rice isolates of M. grisea on the basis of these 65 RAPD bands revealed the presence of high genotypic diversity and continuous DNA fingerprint variation in the pathogen population. No correlation was observed between RAPD patterns and virulence characteristics of the pathogen. The observed population structure contrasted with presumed clonal reproductive behaviour of the pathogen and indicated the possibility of ongoing genetic recombination in the pathogen population. Analysis of the virulence organization of five RAPD groups (RG1–RG5) using 20 rice genotypes comprising at least 15 resistance genes revealed that no combination of resistance genes would confer resistance against all RAPD fingerprint groups present in the M. grisea rice population. The possible implications of the observed population structure of M. grisea for blast resistance breeding have been discussed.     

Two new species of Rhynchosporium

Rhynchosporium consists of two species, R. secalis and R. orthosporum. Both are pathogens of grasses with R. secalis infecting a variety of Poaceae hosts and R. orthosporum infecting Dactylis glomerata. Phylogenetic analyses of multilocus DNA sequence data on R. secalis isolates originating from cultivated barley, rye, triticale and other grasses, including Agropyron spp., Bromus diandrus and Hordeum spp., resolved the monophyletic groups into three species according to their respective hosts. Host specificity according to phylogenetic lineages was confirmed with pathogenicity studies. Because R. secalis was described first on rye this name is retained for Rhynchosporium isolates infecting rye and triticale. Rhynchosporium isolates infecting cultivated barley and other Hordeum spp. and Bromus diandrus belong to a distinct species, R. commune. Similarly isolates infecting Agropyron spp. represent a distinct species of Rhynchosporium, namely R. agropyri. A PCR-RFLP assay was developed as a rapid tool for species identification of R. secalis and R. commune.     

Mating-type Distribution and Fertility Status in Magnaporthe grisea Populations from Argentina

Isolates of Magnaporthe grisea causing gray leaf spot on rice were collected in Argentina and analyzed for mating distribution and fertility. One hundred and twenty-five isolates of M. grisea were collected from rice plants between 2000 and 2003. Each isolate was tested for mating type through a polymerase chain reaction based assay. All M. grisea isolates from Argentina belonged to a single mating type, MAT1.1. The fertility status of isolates was determined using controlled crosses in vitro, pairing each isolate with GUY11 and KA9 (MAT1.2 standard hermaphroditic testers). Production of perithecia was scarce among isolates of the blast pathogen since a low percentage of them (7.2%) developed perithecia with only one of the fertile tester (KA9); all crosses failed with the other tester strain. Asci and ascospores were not observed. The presence of only one mating type and the absence of female fertile isolates indicate that sexual reproduction is rare or absent in M. grisea populations associated with rice in Argentina.     

MAGGY,a retrotransposon in the genome of the rice blast fungusMagnaporthe grisea

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.     

Races of the Barley Root-Knot Nematode,Meloidogyne naasi. I. Characterization by Host Preference

The host preferences of populations of Meloidogyne naasi from England, California, Illinois, Kentucky and Kansas were compared. Among 22 plant species tested, most were hosts for isolates of all five populations; crabgrass was added to the list of known hosts. Differential reactions of isolates on creeping bentgrass, curly dock, sorghum, and common chickweed demonstrated the existence of at least five physiological races within M. naasi. The known races are numerically designated and characterized.     

Mapping of a Magnaporthe grisea locus affecting rice (Oryza sativa) cultivar specificity

Magnaporthe grisea causes rice blast, the most important fungal disease of rice. The segregation of genes controlling virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in this host-parasite interaction. Full-sib progeny and parent isolates Guy11 and 2539 of M. grisea were inoculated onto rice (Oryza sativa) cultivar CO39 and five near-isogenic lines (NILs) of CO39. Each NIL contained a different single gene affecting resistance to specific isolates of M. grisea. No differential interactions between NILs and progeny or parents were observed; parents and progeny pathogenic on CO39 were pathogenic on all five NILs. Segregation ratios of 101 full-sib progeny, 117 progeny from full-sib parents, and 109 backcross progeny, indicated a common single gene affecting pathogenicity on CO39 and the five NILs. A subset of the above 327 isolates (43 fullsib progeny, 37 progeny from full-sib parents, and 32 backcross progeny) were inoculated onto rice cultivar 51583; all were pathogenic, indicating that cultivar specificity to CO39 was segregating in this population of isolates. The locus controlling cultivar specificity, named avrCO39, was mapped to chromosome 1 using a subset of the progeny previously used to construct an RFLP map of M. grisea. The closest reported RFLP markers were 11.8 (estimated 260 kb) and 17.2 cM (estimated 380 kb) away and provide starting points on either side of the locus for a chromosome walk to clone the locus.     

Molecular mapping of two cultivar-specific avirulence genes in the rice blast fungus <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

Rice blast, caused by the fungus Magnaporthe grisea, is a globally important disease of rice that causes annual yield losses. The segregation of genes controlling the virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in the interaction of rice and M. grisea. The segregation of avirulence and virulence was studied in 87 M. grisea F₁ progeny isolates from a cross of two isolates, Guy11 and JS153, using resistance-gene-differential rice cultivars. The segregation ratio indicated that avirulence and virulence in the rice cultivars Aichi–asahi and K59, respectively, are controlled by single major genes. Genetic analyses of backcrosses and full-sib crosses in these populations were also performed. The χ² test of goodness-of-fitness for a 1:1 ratio indicated that one dominant gene controls avirulence in Aichi-asahi and K59 in this population. Based on the resistance reactions of rice differential lines harboring known resistance genes to the parental isolates, two genetically independent avirulence genes, AVR–Pit and AVR–Pia, were identified. Genetic linkage analysis showed that the SSR marker m355–356 is closely linked to AVR–Pit, on the telomere of chromosome 1 at a distance of approximately 2.3 cM. The RAPD marker S487, which was converted to a sequence-characterized amplified region (SCAR) marker, was found to be closely linked to AVR–Pia, on the chromosome 7 telomere at a distance of 3.5 cM. These molecular markers will facilitate the positional cloning of the two AVR genes, and can be applied to molecular-marker-assisted studies of M. grisea populations.     

Genetic Diversity of Magnaporthe grisea in China as Revealed by DNA Fingerprint Haplotypes and Pathotypes

We determined DNA fingerprint haplotypes and pathotypes of the rice‐blast fungus Magnaporthe grisea collected from 13 areas in China. This DNA fingerprinting analysis, using rep‐PCR, of 381 haplotypes (482 isolates) from China indicated that the M. grisea populations cannot be delineated into region‐specific groups. Analyses of the number of alleles (na), Nei's gene diversity, unbiased genetic distance, and Shannon's Information index among 13 populations showed that clusters were not related to the geographic distance between populations with the exception of the Ningxia (NX) and Jilin (JL) cluster. Among northern populations, NX and JL were more similar to one another than to other populations. Pathogen populations consisting of 121 isolates from China were grouped into 53 pathotypes on the basis of disease reaction in differential rice lines. Isolates assayed for pathotypes were detected based on disease reactions. No correlation was observed between fingerprint groups and pathotypes of the pathogen. High frequency of virulence was found on the rice line Shin2 (Pi‐k^s and Pi‐sh) followed by PiNo.4 (Pi‐ta² and Pi‐sh) and K1 (Pi‐ta), while it was low on Kanto 51 (Pi‐k + ?), K3 (Pi‐k^h), and Fujisaka (Pi‐i and Pi‐sh). Virulence was rare on Toride 1 (Pi‐z^t and Pi‐sh). Tetep (Pi‐k^h + ?) was predicted to be a highly effective, as none of the isolates infected this line. These blast‐resistant rice lines can be used in resistance breeding for the effective management of rice blast in the respective regions of China.     

Assessment of Genetic Diversity in Thai Isolates of Pyricularia grisea by Random Amplification of Polymorphic DNA

One hundred and seventy‐four isolates of Pyricularia grisea were collected from various hosts such as barley, rice, weed and wild rice in Thailand. Seven arbitrary decamer primers from the set of University of British Columbia were employed and nine lineages were classified. Lineages B, C and H were predominant, contributing up to 70% of total pathogens in this study. Analysis showed that the distribution of each lineage differs from the predominant lineages across Thailand in such that other lineages were restricted in particular area. For instance, lineage A was limited only in southern Thailand, whereas wide distribution of lineages B and C reflected an influence of both biological and physical effects on pathogen variation. Principal component analysis resulted in a total of four groups of blast pathogen with small distinctions between barley‐, rice‐, weed‐ and wild rice‐infected blast. Bridging relationships occurred among border isolates of weed and rice blast suggesting a chance of migrations between hosts. Higher diversity was observed in northern, north‐eastern and central Thailand while eastern and southern parts were rather low. Genetic diversity indices elucidated an abundance of pathogen lineages existing in northern Thailand suggesting that it should be the centre of diversity.     

Molecular characterization, morphology, and pathogenicity of Alternaria panax from araliaceous plants in Korea

Alternaria blight on araliaceous plants is a common disease caused by Alternaria panax Whetzel and occurs worldwide. Genetic diversity among 58 isolates of A. panax from different araliaceous hosts in Korea was determined by amplified fragment length polymorphism (AFLP) analysis. Selected isolates from genetic groups (determined by AFLP analysis) were examined based on the results of phylogenetic analyses of eight genes (Alt a1, BT1, BT2, EF-1α, gpd, H3, ITS, and RPB2), morphological characteristics, and pathogenicity tests. Isolates were divided into two distinct genetic groups (A and B) by AFLP analysis and based on the results of sequence analyses of the BT1, BT2, gpd, and RPB2 genes. Isolates from ginseng plants (Panax ginseng and P. quinquefolius) fell into group B, whereas isolates from the other araliaceous plants clustered in group A. Morphologically, although some overlap was observed among isolates in the two groups, isolates in group B had longer and narrower conidia, distinct sporulation patterns at 15 °C, and did not secrete pigment into PDA media. Pathogenicity tests revealed that isolates in groups A and B only induced severe disease symptoms on leaves of their hosts, such as isolates in group A on Aralia elata, Aralia continentalis, and in group B on Panax ginseng. The results indicate that the two genetic groups of Alternaria from araliaceous plants should be considered as two different species, and we propose that group A is a candidate of new Alternaria species distinguished from A. panax.     

Genetic Structure of Pyricularia grisea (Cooke) Sacc. Isolates from Italian Paddy Fields

Rice blast disease, caused by the fungus Pyricularia grisea (Cooke) Sacc., is responsible for considerable damages in rice crops in Italy and in other parts of the world. This study was conducted in order to investigate the genetic structure of a P. grisea population in the Po area, the largest rice area in Italy. Rice leaves showing blast symptoms were collected in three successive years (1998–2000) and 43 P. grisea monoconidial culture samples were isolated from infected rice leaves. Fungal DNAs were obtained from mycelia. Moreover, six additional P. grisea DNA samples representative for the five characterized European lineages were also investigated. All 49 DNAs were fingerprinted using the Pot2‐based repetitive polymerase chain reaction specific for the blast pathogen. Unweighted pair‐group method with arithmetic averages cluster analysis shows the presence of three main Italian lineages. Within lineages, similarity was higher than 80%. Samples representative of the three of five known European lineages grouped within these three Italian lineages confirming the presence of three European lineages in Italy. Furthermore, cluster analysis shows the presence of two new haplotypes never found before in the Italian lineage.     

Biosynthesis of secondary metabolites in the rice blast fungus Magnaporthe grisea: the role of hybrid PKS-NRPS in pathogenicity

Fungal secondary metabolites are an important source of bioactive compounds for agrochemistry and pharmacology. Over the past decade, many studies have been undertaken to characterize the biosynthetic pathways of fungal secondary metabolites. This effort has led to the discovery of new compounds, gene clusters, and key enzymes, and has been greatly supported by the recent releases of fungal genome sequences. In this review, we present results from a search for genes involved in secondary metabolism and their clusters in the genome of the rice pathogen, Magnaporthe grisea, as well as in other fungal genomes. We have also performed a phylogenetic analysis of recently discovered genes encoding hybrids between a polyketide synthase and a single non-ribosomal peptide synthetase module (PKS–NRPS), as M. grisea seems rich in these enzymes compared with other fungi. Using results from expression and functional studies, we discuss the role of these PKS-NRPS in the avirulence and pathogenicity of M. grisea.     

The cell death factor,cell wall elicitor of rice blast fungus (Magnaporthe grisea) causes metabolic alterations including GABA shunt in rice cultured cells

An elicitor derived from the cell wall of rice blast fungus (Magnaporthe grisea) causes cell death in suspension cultured cells of rice (Oryza sativa L.). To elucidate the role of M. grisea elicitor on metabolic pathway of rice cells, we performed metabolite profiling using capillary electrophoresis-mass spectrometry (CE/MS). Treatment with M. grisea elicitor increased the amounts of antioxidants and free amino acids and decreased the amount of metabolites in the tricarboxylic acid (TCA) cycle. Lower ATP concentration caused aberrant energy charge, concurrently with reduced amount of NAD(P)H in elicitor treated cells. Among free amino acids detected in this study, the level of gamma-aminobutyric acid (GABA) increased. GABA is metabolized through a bypass pathway of the TCA cycle called GABA shunt, which is composed of glutamate decarboxylase (GAD), GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). While M. grisea elicitor negligibly affected GAD and SSADH, GABA-T activity significantly decreased. The decrease in GABA-T activity was recovered by NADPH oxidase inhibitor, which prevents cell death induced by M. grisea elicitor. Thus, GABA accumulation observed in rice cells under elicitor stress is partly associated with GABA-T activity.Key words: metabolome, Magnaporthe grisea, capillary electrophoresis, mass spectrometry, gamma-aminobutyric acid, GABA transaminase, Oryza sativa     

Nonhost resistance of barley is successfully manifested against Magnaporthe grisea and a closely related Pennisetum-infecting lineage but is overcome by Magnaporthe oryzae

Magnaporthe oryzae is a major pathogen of rice (Oryza sativa L.) but is also able to infect other grasses, including barley (Hordeum vulgare L.). Here, we report a study using Magnaporthe isolates collected from other host plant species to evaluate their capacity to infect barley. A nonhost type of resistance was detected in barley against isolates derived from genera Pennisetum (fontaingrass) or Digitaria (crabgrass), but no resistance occurred in response to isolates from rice, genus Eleusine (goosegrass), wheat (Triticum aestivum L.), or maize (Zea mays L.), respectively. Restriction of pathogen growth in the nonhost interaction was investigated microscopically and compared with compatible interactions. Real-time polymerase chain reaction was used to quantify fungal biomass in both types of interaction. The phylogenetic relationship among the Magnaporthe isolates used in this study was investigated by inferring gene trees for fragments of three genes, actin, calmodulin, and beta-tubulin. Based on phylogenetic analysis, we could distinguish different species that were strictly correlated with the ability of the isolates to infect barley. We demonstrated that investigating specific host interaction phenotypes for a range of pathogen isolates can accurately highlight genetic diversity within a pathogen population.     

Moniliophthora perniciosa,the mushroom causing witches’ broom disease of cacao: Insights into its taxonomy,ecology and host range in Brazil

Witches' broom caused by Moniliophthora perniciosa is the main disease of cacao (Theobroma cacao) in Brazil. The fungus is known to occur on other host families and these populations have been addressed in the literature as biotypes: C (Malvaceae); H (Malpighiaceae); L (Bignoniaceae) and S (Solanaceae). No complete elucidation of the phylogenetic relationships of isolates obtained from this disparate host range appears in the literature. One member of H (ex Heteropterys acutifolia) has been described as a distinct species. But should other biotypes be also recognized as distinct taxa? In the present study, a survey yielding 24 isolates of M. perniciosa from ten hosts and covering a wide range of geographic regions in Brazil was undertaken. These isolates were compared with those from T. cacao using three DNA regions for the phylogenetic analyses: ITS, LSU and RPB1. Morphology was also examined. All isolates in this study were found to belong to M. perniciosa, including the population from H. acutifolia, formerly treated as Moniliophthora brasiliensis but reduced here to a synonym of M. perniciosa. This species ranged from pathogenic to a previously unreported occurrence as a non-pathogenic endophyte in the Atlantic rainforest tree Allophylus edulis (Sapindaceae). M. perniciosa was recorded on a range of solanaceous hosts (16 species) over a wide variety of ecosystems. The ecological and evolutionary significance of these novel findings are discussed.     

A Rho3 homolog is essential for appressorium development and pathogenicity of Magnaporthe grisea

The small GTPase Rho3 is conserved in fungi and plays a key role in the control of cell polarity and exocytosis in yeast. In this report, we show that a Rho3 homolog, MgRho3, is dispensable for polarized hyphal growth in the rice blast fungus Magnaporthe grisea. However, MgRho3 is required for plant infection. Appressoria formed by the Mgrho3 deletion mutants are morphologically abnormal and defective in plant penetration. Conidia of the Mgrho3 deletion mutants are narrower than those of the wild-type strain and delayed in germination. Transformants expressing a dominant negative Mgrho3 allele exhibit similar phenotypes as the Mgrho3 deletion mutant, while transformants expressing a constitutively active allele of MgRho3 can produce normal conidia but remain defective in appressorium formation and plant infection. In contrast, overexpression of wild-type MgRho3 increases the infectivity of M. grisea. Our results reveal a new role for the conserved Rho3 as a critical regulator of developmental processes and pathogenicity of M. grisea.     

Enhancement of disease resistance to Magnaporthe grisea in rice by accumulation of hydroxy linoleic acid

Linoleic acid (18:2) and linolenic acid (18:3) are sources for various oxidized metabolites called oxylipins, some of which inhibit growth of fungal pathogens. In a previous study, we found disease resistance to rice blast fungus Magnaporthe grisea enhanced in 18:2-accumulating transgenic rice (F78Ri) in which the conversion from 18:2 to 18:3 was suppressed. Here, we demonstrate that 18:2-derived hydroperoxides and hydroxides (HPODEs and HODEs, respectively) inhibit growth of M. grisea more strongly than their 18:3-derived counterparts. Furthermore, in F78Ri plants, the endogenous levels of HPODEs and HODEs increased significantly, compared with wild-type plants. These results suggest that the increased accumulation of antifungal oxylipins, such as HPODEs and HODEs, causes the enhancement of disease resistance against M. grisea.     

稻瘟病菌拮抗性大豆根瘤内生细菌的筛选及抑制效果

【目的】从河南大豆根瘤的内生细菌资源中筛选对稻瘟病菌有拮抗作用的菌株,初步探讨其抑菌效果,为进一步研究其抑菌机理提供菌种资源。【方法】以稻瘟病菌为供试病原菌,采用对峙法进行拮抗性菌株筛选,显微观察法研究受抑制病原菌菌丝变化,对筛选拮抗性菌株进行细胞形态学及生理生化特性试验、16S rRNA基因测序和系统发育分析及接种防效试验。【结果】经复筛有17株内生菌拮抗效果较明显,最高抑制率为62.16%;受抑制病原菌丝呈现弯曲打结、断裂、原生质浓缩等畸形状态。拮抗性筛选过程中内生菌快速生长形成生物薄膜,包埋菌丝并使其断裂。拮抗菌株分布在7属9种,稻瘟病拮抗性大豆根瘤内生菌呈现种属多样性。防效试验表明内生菌处理组稻苗发病率和病情指数均显著降低,防治效果最高达74.19%。【结论】大豆根瘤内生拮抗性菌株具有种属多样性,拮抗性菌株处理组稻苗发病率和病情指数均显著降低,防治效果显著,为进一步研究其抑菌机理提供菌种资源。