Geographic Distribution of Mating Types in Magnaporthe grisea and the Relationship Between Fertile Isolates in China

377 isolates of Magnaporthe grisea were collected from 17 provinces in China and their geographic distribution of mating types and their fertility was tested with four standard isolates, KA3 and TH12 (Mat1.1) and Guy11 and TH16 (Mat1.2) provided by CIRAD. 73 fertile isolates were tested with SCAR markers of 13 pairs of primers. Preliminary results showed that the geographic distribution of M.grisea existed among isolates collected from the same location as well as different locations and the genetic relationship between fertile isolates of the fungus in China. The existence of sexual reproduction of M .grisea was explored in the field as well.

Analysis of the Abnormal Segregation of Pathogenicity in Magnaporthe grisea by Using a Genetic Cross of Oryza and Eleusine Isolates

A genetic cross between Oryza isolate Y93-164a-1 and Eleusine isolate SA98-4 was established, and the pathogenicity of 151 F1 progeny isolates was investigated on both host plants rice and finger millet. Results showed that the segregation of pathogenicity in this genetic cross was abnormal, i.e., most of the progeny isolates were nonpathogenic on both host plants. However, no abnormal segregation was observed when middle repetitive sequence MGR586 and 31 single-copy RFLP markers from all of the chromosomes were genetically analyzed. At the same time, comparison of the chromosomal organization among two pairs of parental isolates did not find any genomic abnormity. These results suggested that the ＂abnormal＂ inheritance of pathogenicity in this cross was most likely due to the reassortment of numerous host species specificity genes but not the biased segregation of the host species specificity genes. The host species specificities in M. grisea were likely to be multigenically controlled, at least in the genetic cross involving rice pathogen and the grasses pathogen other than rice.

Resistance Evaluation of Some Chinese Leading Rice Maintainer, Restorer lines and Their Hybrids to Magnaporthe grisea

Six isolates of Magnaporthe grisea were selected to inoculate on 10 Chinese leading maintainer lines (B-lines), 14 restorer lines (R-lines) and their F1 hybrid plants. In the tested rice materials, R-lines were proved to be more resistant to blast than B-lines. The resistance frequency of about 25% F1 hybrid plants was less than their parents. In addition, 26 isolates of M. grisea collected from different rice growing areas of China were inoculated on 13 new improved hybrid rice combinations. The resistance frequencies of 5 improved hybrids were better than those of the controls and leading varieties in rice production of China.

Expression of a Magnaporthe grisea Elicitor and Its Biological Function in Activating Resistance in Rice

The expression of a protein elicitor from Magnaporthe griesea and its biological function in activating resistance in rice （Oryza safiva L） were reported. The gene of elicitor was expressed in Escherichia colicells and produced a His6-fusion protein with 42 kD apparent molecular weight on SDS-PAGE. The purified protein could induce the resistance to blast disease, with the control efficiency of 46.47% and 36.41% at the 14^th day and the 21^st day after blast inoculation, respectively. After treatment with the expressed protein, the phenylalanine ammonia-lyase （PAL） and peroxidase （POD） activities were promoted in rice plants, meanwhile, the transcription levels of STKM, FAD, PBZ1 and PR1 genes were increased in rice plants. Moreover, after comparing the profile of total rice leaf proteins on two-dimensional electrophoresis gel, about 14 proteins were found to be increased in expression level after the expressed protein treatment. All the results indicated that the expressed protein could act as an elicitor to trigger the resistance in rice.

Sensitivity Detection Technique and Resistance Risk Assessment of Magnaporthe grisea to Tricyclazole

In vitro detection method for the sensitivity of Magnaporthe grisea to tricyclazole was studied, and the potential resistance risk of blast disease to tricyclazole was assessed. Both EC50 of hyphal melanization (EC50-H) and minimum inhibitive concentration of melanization in appressorial (MIC-A) by inhibitor tricyclazole showed positive correlation to the EC50 of tricyclazole against blast disease tested in vivo, with relative co-efficiency (R5) of 0.8995 and 0.8244, respectively. However, stability and reproducibility of EC50-H were better than those of MIC-A, suggesting that it could be used to detect the sensitivity of M. grisea to tricyclazole in vitro. Tricyclazole sensitivity of the progenies derived from single spores of the most sensitive isolate DY2 and the least sensitive isolate GY6 detected in sensitivity monitoring in 2000 was not stable, with mean EC50 values of 4.4968 μg/mL and 5.4010 ug/mL, respectively, indicating that the difference in EC50 between DY2 and GY6 was not caused probably by resistance variation. EC50 of GY6 did not increase significantly when continuously selected for twenty generations under the selection pressure of tricyclazole in vivo. However, the sensitivity of DY2 was decreased by 10-fold after selected for twenty generations. The results suggested that tricyclazole was still low resistance risk for M. grisea in China.

Physiological Macro-lesions Enhanced Resistance to Blast (Magnaporthe grisea) in Rice Near-isogenic Lines

Roll-leaf-1 （rl-1） and spot-leaf-1 （spl-1） were two near-isogenic lines, which were obtained after 3 to 4 backcrosses with early season indica rice Zhefu 802 as recurrent parent. Henna macro-lesions, referred as physiological or morphological markers, began to appear on leaves at 4.5- to 6.0-leaf stage. The rice seedlings were inoculated at 3.5-, 5.0- and 7.0-leaf stages with high pathogenic races Zhong A1 and Zhong B1 of Magnaporthe grisea, respectively. The resistance of rl-1, spl-1 and Zhefu 802 against blast was significantly different. The seedlings of Zhefu 802 at 3.5- to 7.0-leaf stage were susceptible to races Zhong A1 and Zhong B1 of M. grisea, whereas those of rl-1 and spl-1 at 3.5-, 5.0- and 7.0-leaf stages were susceptible, moderately resistant and resistant, respectively. These results suggested that the enhanced resistance of rl-1 and spl-1 related to the appearance of their morphological marker lesions. The experiment provided a basis for studying lesion mimic and hypersensitive response in association with disease resistance.

Factors affecting appresorium formation of Magnaporthe grisea

M. grisea causes one of the major diseases on rice, rice blast. Appresoriumformation is the key for the pathogen to penetrate into host tissue. So, it is im-portant to study the effect of environmental factors on the appresorinm formation

Mating Type Alleles, Female Fertility and Genetic Diversity of Magnaporthe grisea Populations Pathogenic to Rice fromSome Asian Countries

Five hundred and twenty-two isolates of Magnaporthe grisea isolated from rice in 5 Asian countries were characterized for their mating type by crossing them with 4 hermaphroditic isolates(KA3 and TH12: MAT1.1; Guy11 and TH16: MAT1. 2). Among them, 41% were MAT1.1 and 25% were MAT1. 2. The remaining 34% did not produce perithecia with any of the 4 hermaphroditic testers. In Bangladesh, India, Nepal, Vietnam and in most provinces of China, both mating types were present. Only one mating type was found in 3 provinces and 1 city of China. Almost all the isolates had very low fertility, as they were in general female sterile and sometimes also male sterile. Hermaphroditic isolates were recovered from the 5 countries. In these countries, they represented between 13% and 75% of the isolates. In Zhejiang, Guizhou, Guangdong, Hunan, Yunnan and Hubei provinces of China, hermaphroditic isolates represented between 6% and 67%. The genetic diversity of 143 isolates from these countries and provinces, where hermaphroditic isolates had been collected, was analyzed using SCAR markers. Genetic diversity was high and population structure did not resemble classical clonal structure described in most rice growing regions. The existence of sexual reproduction in the field, localization of a center of diversity in China, and migration between countries were discussed in this paper.

Resistance Evaluation of Some Hybrid Rice, Conventional Early Indica and Late Japonica Rice to Magnaporthe grisea

Thirty isolates of Magnaporthe grisea collected from 18 provinces/cities representing 21 pathotypes and 9 different lineages were inoculated to rice varieties with known resistance genes and some hybrid rices, conventional early indica and late japonica varieties cultivated recently in China. Virulence spectrum of the 30 isolates was very different, showing that they recognize numerous different resistance genes. Varieties also revealed very different resistance patterns showing that they carry different resistance genes or combinations of resistance genes. On the basis of comparisons with international differential varieties with known resistance genes, resistance genes in certain Chinese varieties could be speculated. The results indicated that some of them were resistant to most of the isolates tested and that they could be of interest as resistance sources for hybrid parents or to be planted in the field directly.

Promoter trapping in Magnaporthe grisea

Application of promoter trapping based on transformation in Magnaporthe grisea is reported in this paper. Two promoter-trapping vectors, designated as pCBGFP and pEGFPHPH, were constructed and transformed into protoplasts of M. grisea. A library of 1077 transformants resistant to hygromycin B was generated. Of which, 448 transformants were found to express eGFP gene in different structures of M. grisea. Three transformants grew slowly, 5 transformants decreased in conidiation and 7 transformants reduced in pathogenicity greatly among these 448 transformants. Eleven transformants were checked by genomic southern blot randomly, and 9 of which were single-copy insertions. The promoter trapping technique has been applied successfully in M. grisea and can be used as a tool for functional genomic analysis.

Recovery and Biological Properties of Nitrate Non-utilizing Mutants of Rice Blast,Magnaporthe grisea

Eleven nitrate non-utilizing (nit) mutants were recovered from six isolates of Magnaporthe grisea cultured on MM media -amended with 60 g/L potassium chlorate, with a frequency of 1.42 %. Some biological properties, such as growth rate, growth biomass, cultural characters, conidial production, sexual reproduction ability, and pathogenicity were compared between nit mutants and their parent isolates. Results showed that all the nit mutants were resistant to chlorate. Some important biological properties such as the growth rate on YPSA, conidial production ability on TPSA, pathogenicity, had no significant differences between nit mutants and their parent isolates. Mating type didn't change, but perithecia production ability of fertile isolates changed significantly as compared with that of their parent isolates. Therefore, the nit can be used as a genetic marker to study the genetics such as pathogenicity, fungicide resistance in Magnaporthe grisea.

Structure and Expression of Several Putative Cdc42-Interacting Proteins in Magnaporthe grisea

MgCdc42 （Cdc42 in Magnaporthe grisea）, with high homology to ScCdc42 （Cdc42 in Saccharomyces cerevisiae）, has been demonstrated to involve in the morphogenesis and infection process. To further understand the signaling network, the putative MgCdc42-interacting proteins were analyzed. ScCdc42-interacting protein sequences were first used to BLAST against the M. grisea genome database to retrieve their corresponding analogs. Subsequently, conserved domains of these proteins were compared and expression patterns of their encoding genes in different MgCdc42 mutation states were analyzed by semiquantitative RT-PCR. All retrieved analogs of ScCdc42-interacting proteins from the M. grisea database have conserved domains as those in S. cerevisiae. Expression of their encoding genes increased in MgCdc42CA mutant and decreased in MgCdc42KO mutant. However, MgBeml, Chin1, and MgGicl in MgCdc42DN mutant had the same expression level as that in the wild type, although MgBem4, MgBoi2, MgCdc24, MgGic2, MgRgal, and Mst20 had decreased expression level, as expected. Overall, it is concluded that there may exist a similar Cdc42 signal pathway in M. grisea as in S. cerevisiae and MgCdc42 plays a key role in the pathway.

Vegetative Compatibility and Asexual Recombination in Magnaporthe grisea from Jiangsu Province, China

Vegetative compatibility among isolates of different races in Magnaporthe grisea collected from Jiangsu Province and asexual recombination among compatible isolates by anastomosis were tested. Twenty isolates involving seven races from diseased rice plants were paired on polished rice rose bengal medium and incubated at 25℃ in darkness for 18 days. Among 173 pairings tested, solid hyphal fusion lines formed by anastomosis between 124 pairings, indicated that these isolates were vegetative compatible with each other. The result showed that most M. grisea isolates were vegetative compatible. Furthermore, 17 vegetative compatible pairings between monoconidial isolates with MBCsIPTr marker and isolates with MBCrIPTs marker were selected to detect the asexual recombination between the compatible isolates of different races. The asexual recombinants with MBCrIPTr marker were detected in single hyphal fragment progenies in thirteen of the seventeen pairings. The percentage of recombinants was about 0. 6 -11.3%. Results showed that vegetative compatibility was prevailing among isolates of M. grisea in Jiangsu Province in vitro. These results also suggested that asexual recombination may be an important mechanism for M. grisea to maintain genetic diversity in nature.

Isolating cDNA clones from rice induced by Magnaporthe grisea using PCR based differential screening method

The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that

In silico Analysis of the Potential Infection Mechanisms of Magnaporthe grisea from Horizontal Gene Transfer Hypothesis

Horizontal gene transfer （HGT） has long been considered as a principal force for an organism to gain novel genes in genome evolution. Homology search, phylogenetic analysis and nucleotide composition analysis are three major objective approaches to arguably determine the occurrence and directionality of HGT. Here, 21 genes that possess the potential to horizontal transfer were acquired from the whole genome of Magnaporthe grisea according to annotation, among which three candidate genes （corresponding protein accession numbers are EAA55123, EAA47200 and EAA52136） were selected for further analysis. According to BLAST homology results, we subsequently conducted phylogenetic analysis of the three candidate HGT genes. Moreover, nucleotide composition analysis was conducted to further validate these HGTs. In addition, the functions of the three candidate genes were searched in COG database. Consequently, we conclude that the gene encoding protein EAA55123 is transferred from Clostridium perfringens. Another HGT event is between EAA52136 and a certain metazoan＇s corresponding gene, but the direction remains uncertain. Yet, EAA47200 is not a transferred gene.

Induced Pib Expression and Resistance to Magnaporthe grisea are Compromised by Cytosine Demethylation at Critical Promoter Regions in Rice

Pib is a well-characterized rice blast-resistance gene belonging to the nucleotide binding site （NBS） and leucine-rich repeat （LRR） superfamily. Expression of Pib was low under non-challenged conditions, but strongly induced by the blast-causing fungal pathogen Magnaporthe grisea, thereby conferring resistance to the pathogen. It is generally established that cytosine methylation of the promoter-region often plays a repressive role in modulating expression of the gene in question. We report here that two critical regions of the Pib promoter were heavily CG cytosine-methylated in both cultivars studied. Surprisingly, induced expression of Pib by M. grisea infection did not entail its promoter demethylation, and partial demethylation by 5-azacytidine-treatment actually reduced Pib expression relative to wild- type plants. Accordingly, the blast disease-resistance was compromised in the 5＇-azaC-treated plants relative to wild-type. In contrast, the disease susceptibility was not affected by the 5＇-azaC treatment in another two rice cultivars that did not contain the Pib gene, ruling out effects of other R genes and non.specific genotoxic effects by the drug-treatment as a cause for the compromised Pib-conditioned blast-resistance. Taken together, our results suggest that promoter DNA methylation plays a novel enhancing role in conditioning high-level of induced expression of the Pib gene in times of M. grisea infection, and its conferred resistance to the pathogen.

Pathogenicity Analysis on Magnaporthe oryzae from Hybrid Combination Wuyou 308

Eighteen blast isolates were obtained from hybrid combination Wuyou308 using the Magnaporthe oryzae pathogen isolation method.Race identification of these isolates was conducted based on seven Chinese blast differentials and 11 blast monogenic lines.The results indicated that the isolates were identified as the races of ZB13,ZB15 and ZC13,accounting for 66.67%,27.78%,5.56%,respectively,and the resistance genes including Pi-ta2 and Pi-sh,Pi-i were highly susceptible to these isolates,while the resistance genes like Pi-kh,Pi-1,Pi2,Pi-9 and Pi-50 showed good resistance to tested pathogens.All isolates were compatible to the original rice hybrid Wuyou308.Three isolates including GDHY-308-1401 were used for testing their pathogenicity to 45 local varieties.The results demonstrated that 13 varieties appeared highly susceptible to the tested isolates,accounting for 28.89%;two varieties appeared moderately susceptible to the tested isolates,accounting for 4.44%;30 varieties showed moderately/highly resistance,accounting for 66.67%.Among them,some of new hybrid combinations such as Wufengyou 9802,Wuyou 613,Wuyou 1179 showed good resistance to the inoculated strains,and they were recommended to be candidates in the rice region where Wuyou308 showed susceptibility.

Host Active Defense Responses Occur within 24 Hours after Pathogen Inoculation in the Rice Blast System

Phenotypical, cytological and molecular responses of rice to the fungus Magnaporthe grisea were studied using rice cultivars and lesion mimic plants. The cultivar Katy was susceptible to several virulent M. grisea isolates, and a Sekiguchi like-lesion mimic mutant of Katy （LmmKaty） showed enhanced resistance to these isolates. Lesion mimic phenotype of LmmKaty was rapidly induced by virulent M. grisea isolates or by avirulent ones only at high levels of inoculum. Autofluorescence （a sign of an active defense response） was visible under ultraviolet light 24 h after localized inoculation in the incompatible interaction, whereas, not evident in the compatible interaction. Autofluorescence was also observed in LmmKaty 20 h after pathogen inoculation, indicating that rapid cell death is a mechanism of LmmKaty to restrict pathogen invasion. Rapid accumulations of defense related （DR） gene transcripts, phenylalanine ammonia lyase and β-glucanase, were observed beginning at 6 h and were obvious at 16 h and 24 h after inoculation in an incompatible interaction. Rapid transcript accumulations of PR-1 and chitinase had occurred by 24 h after inoculation in an incompatible interaction. Accumulations of these transcripts were delayed in the compatible interaction. These results indicate that host active defense responses occur 24 h after pathogen inoculation and that LmmKaty exhibits enhanced resistance to M. grisea. It is suggested that the autofluorescence and expression of the DR genes after heavy inoculation are important cytological and molecular markers respectively for early determination of the host response to M. grisea in the rice blast system.

Evaluation of 0.3% Agricultural Antibiotic 702 AS against Three Pathogenic Fungi of Rice

Abstractsolution （AS） against Rhizocto- nia solani, Magnaporthe grisea and Ustilaginoidea virens. [ Method] The toxicity effect of 0.3% agricultural antibiotic 702 AS against R., solani, M. grisea and U. virens were studied by mycelial growth rate method and mycelial wet weight method. [ Result ] The EC50 and EC50 values of agricultural antibiotics 702 AS to R. solani, M. grisea and U. virens in vitro condition were 4.16 and 16.17 mg/L, 16.05 and 41.85 mg/L, 111.2 and 389.0 mg/L, respectively. Agricultural antibi- otics 702 AS had good inhibition effect against R. solani and M. grisea and could basically achieve the effects of commercially available Jinggangmycin and kaanga- mycin, but the inhibition effect against U. v/rens was relatively weak. The preventive activity of aqueous solution was obviously higher than treatment activity. [Conclusion] 0.3% Agricultural antibiotic 702 AS is considered to be a potential biological control agent against R. solani, M. grisea and U. virens, and can be used as the potential alternative pesticide of Jinggangmycin.