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.

Identification of a Lectin Gene Induced in Rice in Response to Magnaporthe grisea Infection

By mRNA differential display, eight induced cDNAs were obtained from rice leaves infected with an incompatible race 131 of Magnaporthe grisea, and one of these cDNAs was highly similar to salt-induced mannose-binding lectin gene. Using this fragment as a probe, a full length cDNA was isolated from a nice cDNA library, which was constructed using mRNA from the incompatible race-infected leaves. Sequence analysis indicates that the cDNA encodes a protein of 15 kD with 145 amino, acids and shares 96% identity at nucleotide level with MRL and salT, but is identical to MRL at amino acid level. Genomic Southern blotting shows that there are two mannose-binding lectin genes in rice genome. Northern blotting analysis indicates that the gene was strongly and specifically induced in rice leaves infected with the incompatible race, suggesting that the lectin induction be involved in the defense of rice to M. grisea.

UV-B辐射增强对水稻稻瘟病菌(Magnaporthe grisea)生长和产孢的影响

采用人工紫外灯照射方法,研究了在UV-B辐射增强条件下和UV-B不同剂量作用下,水稻稻瘟病菌(Mag-naporthe grisea)菌丝的生长速率、菌落直径、菌落形态、菌丝形态及产孢量。结果表明,UV-B辐射增强可延缓稻瘟病菌的生长,导致菌丝生长速率和产孢量均显著下降(P<0.05)。同一UV-B辐射强度下,不同的辐射时间对稻瘟病菌菌丝生长速率和菌落直径的影响没有显著性差异(P>0.05)。在2.5和5.0 kJ.m-2强度下,不同的辐射剂量对病菌产孢量的影响有显著性差异(P<0.05)。在UV-B辐射增强条件下,病菌采取与植物类似的形态学变化来抵御UV-B辐射增强的伤害,经UV-B辐射处理后的病菌菌落颜色加深,菌丝分布更加致密。

稻瘟病菌(Magnaporthe grisea)诱导的水稻早期反应基因ER1的5’端cDNA片段克隆及序列分析

The 5’ end cDNA fragment(ER1’) of early responsive gene(ER1) of rice(Oryza sativa L. subsp. japonica No.4) induced by blast fungus M. grisea has been isolated and sequenced (606 bp) by 5’RACE technique(Fig. 2). DNA sequence analysis showed that ER1’ (606 bp) can encode 134 amino acids and there are 309 bp nucleotides located at the 3’ end of the untranslated region of mRNA of ER1 gene(Fig. 3). The 89 bp overlapping fragment was found between the 5’ end of ER1 cDNA fragment and the 3’ end of ER1’(Fig. 3). The partial identities of amino acid sequence deduced from ER1’ with an unknown protein(gene accession number AC002409) of Arabidopsis thaliana was 82%(Fig. 4).

Representative appressorium stage cDNA library of Magnaporthe grisea

A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a λTriplEx2 vector by SMART?cDNA library containing 2.37×106 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M grisea in GenBank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M grisea.

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.

Race Specificity of Major Rice Blast Resistance Genes to Magnaporthe grisea Isolates Collected from indica Rice in Guangdong, China

Race-specific resistance and field resistance of 30 rice blast resistance monogenic lines derived from different resources were evaluated. The spectra of resistance to 163 Magnaporthe grisea isolates collected from indica rice in Guangdong Province, China ranged from 0.6% to 89.6%. Most of the monogenic lines showed a narrow resistance spectrum and high susceptibility in rice blast area, whereas the lines with Pikh and Pi1(t) had the broad resistance spectra of 89.6% and 82.2% respectively, showing a high and stable blast resistance in fields. According to the cluster analysis of specific resistance to 163 blast isolates tested, the 30 monogenic lines were divided into 15 groups, and based on the principal factor analysis, nine kinds of race-specific resistance were identified. Pik, Piz5, Pi9 and Pish can be used as candidate resistance genes for rice breeding since their specific resistance differed from those of the backbone parents in Guangdong, China. Gene pyramiding of Pikh [or Pi1(t)], Pi9 (or Piz5) and Pish (or Pita2) will be effective to obtain broad-spectrum blast resistance in rice breeding program in Guangdong, China. The strategies for studying and application of rice blast resistance genes were discussed.

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.

Application of cDNA array for studying the gene expression profile of mature appressoria of Magnaporthe grisea

Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles ofappressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST （expressed sequence tag） database ofM. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTHll, beta subunit of G protein and SGTI involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results.

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.

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.

Cloning,sequencing and expression analysis of the NAR promoter activated during hyphal stage of Magnaporthe grisea

The promoter of NAR gene in Magnaporthe grisea was isolated and sequenced. The promoter sequences contained the "TATA" box, the "CAAT" box, and binding sites for fungal regulatory proteins. Programs that predict promoter sequences in-dicated that promoter sequence lies between locations 430 and 857 of the NAR promoter fragment. GFP expression under the NAR promoter and NAR transcript analysis revealed that this promoter is activated primarily at the mycelial stage in the rice blast fungus and could be used to express native or extrinsic genes in the mycelia of the rice blast fungus.

Sequence analysis and expression pattern of MGTA1 gene in rice blast pathogen Magnaporthe grisea

MGTA1, a putative fungal Zn（Ⅱ）2Cys6 transcriptional activator-encoding gene, was isolated from rice blast pathogen Magnaporthe grisea, which is homologous to CLTA1 from Colletotrichum lindemuthianum with 51% identity at protein level. MGTA1 cassette contains a 2370 bp open reading frame, consisting of 6 exons, coding a 790 amino acid peptide. MGTA1 gene exists as a single copy in genomes of 7 strains of M. grisea, and is expressed in tip hyphae, conidia, and mature appressoria of strain Guy 11.

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.

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.

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.

Pathotype Structure of Magnaporthe grisea in the Fields of Chongyang and Yuan'an in Hubei Province,China

[Objective] This study aimed to explore the pathotype structure of Magnaporthe grisea in Chongyang and Yuan'an in Hubei Province,China.[Method] From the rice-growing fields of Chongyang and Yuan'an in Hubei Province where rice blast occurs frequently,60 isolates which were pathotyped against two sets of host differentials:Chinese host differentials and CO39 NILs,were obtained.Then,20 pathotypes with the six indica host differentials(CO39 NILs) were observed,while 13 pathotypes in four race groups were observed out of the same single spore isolates with Chinese host differentials which consists of three indica cultivars and four japonica cultivars.The diversity of the pathotypes of M.grisea populations tested by CO39 NILs was 2.54 and the pathotype 137.1 occurred at predominantly high frequency(21.67%).The diversity of physiological races of M.grisea populations tested by Chinese host differentials was 1.22 and the race group ZA occurred at predominantly high frequency(73.33%).The diversity of physiological races of M.grisea in Chongyang and Yuan'an were also calculated.Overall,the diversity of pathotypes of M.grisea in Yuan'an was higher than that in Chongyang with the two sets of the host differentials.[Conclusion] This study provided current information on the pathotype spectrum of M.grisea populations in the rice fields of Hubei Province to allow the formulation of viable strategies for blast resistance breeding programs in Hubei Province.

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.