Evaluation and Genetic Classification of Rice Germplasm Resources Resistant to Rice Blast

[ Objective] This study aimed to explore rice germplasm resources resistant to rice blast and provide theoretical basis for the modification of rice blast- resistant germplasm resources. [ Method] The resistance of 120 rice materials to rice blast was identified at three experimental areas. [ Result] The resistance of 120 experimental materials was at grade 0 -9 to panicle blast in Hainan Province, at grade 0 -9 to seedling blast in Fujian Province and at grade 3 -9 topanicle blast in Guangxi Zhuang Autonomous Region. Analysis of correlations between agronomic traits and rice blast resistance indicated that the resistance of experimental materials could not be simply determined based on agronomic traits. Furthermore, 61 published SSR makers closely linked to rice blast were selected and synthe- sized for genotypic analysis of 120 experimental materials. The results of cluster analysis preliminarily divided 120 germplasms into resistant varieties and susceptible varieties. [ Conclusion] These rice germplasm resources are of important value for breeding new materials resistant to rice blast.

Analysis of Resistant Spectrum to Rice Blast in Transgenic Rice Lines Introduced Lysozyme Gene from T4 Phage

The receptor cultivar Nan29 and thirty-six T5 rice lines derived from ten T0 generation transgen-ic plants harboring lysozyme gene were challenged in the greenhouse by inoculating 63 isolates belonging to 48 races of Magnaporthe grisea from Yunnan Province. The transgenic rice lines exhibited resistance to more than 72% of isolates inoculated in this experiment, and 38.1% (24 isolates) of them could infect the receptor cultivar Nan29. The results indicated that the transgenic rice lines possessed wide-spectrum resistance against various rice blast races and the resistant spectrum of rice lines were different although some lines derived from same T0 plant. The transgenic rice lines exhibited also high resistance to leaf and neck blast in the disease field evaluation, but not all of resistant lines against leaf blast were resistant to neck blast.

Development and Identification of Novel Rice Blast Resistant Sources and Their Characterization Using Molecular Markers

To develop and characterize introgression lines for leaf and neck blast resistance, 326 introgression lines were developed using various accessions of six different AA genome wild species in the genetic background of elite Indian varieties like PRl14 and Pusa 44 and were screened for blast resistance. Stringent phenotyping coupled with genotyping using gene based markers led to the identification of four resistant introgression lines, which showed promising resistance and do not possess any of the tested genes. Furthermore, multi-location screening confirmed the field resistance of the four introgression lines to both leaf and neck blast. Molecular characterization of these introgression lines using genome-wide simple sequence repeat markers revealed the presence of small percentage of wild Oryza genome introgrssion. So these lines can be used for mapping and identification of novel leaf and neck blast resistance genes. Thus, these four introgression lines can be considered as new genetic resources for blast resistance.

Strategy for Use of Rice Blast Resistance Genes in Rice Molecular Breeding

Rice blast is one of the most destructive diseases affecting rice production worldwide.The development and rational use of resistant varieties has been the most effective and economical measure to control blast.In this review,we summarized the cloning and utilization of rice blast resistance genes,such as Pi1,Pi2,Pi9,Pi54,Pigm and Piz-t.We concluded that three main problems in the current breeding of rice blast resistance are:availability of few R(resistance)genes that confer resistance to both seedling and panicle blast,the resistance effect of pyramided lines is not the result of a simple accumulation of resistance spectrum,and only a few R genes have been successfully used for molecular breeding.Therefore,novel utilization strategies for rice blast R genes in molecular breeding were proposed,such as accurately understanding the utilization of R genes in main modern rice varieties,creating a core resistant germplasm with excellent comprehensive traits,screening and utilizing broadspectrum and durable resistance gene combinations.Lastly,the trends and possible development direction of blast resistance improvement were also discussed,including new genes regulating resistance identified via GWAS(genome-wide association study)and improving rice blast resistance using genetic editing.

Resistance Evaluation of Dominant Varieties against Rice Blast (Magnaporthe oryzae) and Bacterial Blight (Xanthomonas oryzae pv. oryzae) in Guangzhou, China

The resistance of dominant rice varieties in Guangzhou against rice blast(Magnaporthe oryzae)and bacterial blight(Xanthomonas oryzae pv.oryzae)were evaluated by artificial inoculation and field natural blast nursery identification.Among 11 varieties tested,seven varieties showed resistance to rice blast,accounting for 63.6%;two varieties were highly resistant,accounting for 18.2%;three varieties were resistant,accounting for 27.3%;two varieties were moderately resistant,accounting for 18.2%;four varieties were susceptible(moderately susceptible),accounting for 36.4%.Varieties showed different resistance against the prevailing pathotype IV of bacterial blight;one variety was resistant,accounting for 9.1%;three varieties were moderately resistant,accounting for 27.3%;five varieties were moderately susceptible,accounting for 45.5%;one variety was susceptible,accounting for 9.1%;one variety was highly susceptible,accounting for 9.1%;all varieties tested showed susceptible to the virulent pathotype V.Huanghuazhan,Yuejingsimiao 2 and Fengxiuzhan showed resistance against both bacterial blight(pathotype IV)and rice blast,accounting for 27.3%.It is helpful to reduce the production risk by extending such dominant rice varieties with good resistance to rice blast and bacterial blight.

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety （Digu） by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 （pZH01-tp309） and three different expression constructs, pCB-Pi-d25.3kb （pCB5.3kb）, pCB-Pi-d26.3kb （pCB6.3kb） and pZH01-Pi-d22.72kb （pZH01-2.72kb） of Pi-d2, driven by Pi-d2 gene’s own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium- mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained.

The OsBSK1-2-MAPK module regulates blast resistance in rice

Receptor-like cytoplasmic kinase OsBSK1-2 was reported to play an important role in regulation of response to rice blast,but the signaling pathway remained unknown.In this study,we identified OsMAPKKK18 and previously uncharacterized MAPKKKs OsMAPKKK16 and OsMAPKKK19 that interact with OsBSK1-2.Expression of all three MAPKKKs was induced by Magnaporthe oryzae infection,and all three induced cell death when transiently expressed in Nicotiana benthamiana leaves.Knockout of OsMAPKKK16 and OsMAPKKK18 compromised blast resistance and overexpression of OsMAPKKK19 increased blast resistance,indicating that all three MAPKKKs are involved in regulation of rice blast response.Furthermore,both OsMAPKKK16 and OsMAPKKK19 interacted with and phosphorylated OsMKK4 and OsMKK5,and chitin-induced MAPK activation was suppressed in osmapkkk16 and osbsk1-2 mutants.OsMAPKKK18 was earlier reported to interact with and phosphorylate OsMKK4 and affect chitin-induced MAPK activation,suggesting that OsBSK1-2 is involved in regulation of immunity through multiple MAPK signaling pathways.Unlike BSK1 in Arabidopsis,OsBSK1-2 was not involved in response to avirulent M.oryzae strains.Taken together,our results revealed important roles of OsMAPKKK16/18/19 and a OsBSK1-2-OsMAPKKK16/18/19-OsMKK4/5 module in regulating response to rice blast.

Molecular Detection of Rice Blast Resistance Genes in Major Varieties of Rice in Jilin Province

[ Objective] This study aimed to detect flee blast resistance genes in major varieties of rice in Jilin Province. [ Method ] Ten molecular markers closely linked with rice blast resistance genes were used for molecular identification of resistance genes in 24 rice varieties from Jilin Province. [ Result] The 24 major vari- eties of rice widely lacked in rice blast resistance genes Pib, Pil, Pikh and Pia; Pi9 gene was absent in 17 of the 24 major varieties, especially in Jijing 88 and Changbai 9 which occupied the largest cultivation area in Jilin Province ; in addition, Jijing 88 harbored five rice blast resistance genes, including Pita, Pikm, Pt2, P/-d2 and P/z. [ Conclusion] Pib, Pil, Pikh, Pia and Pi9 are the major resistance genes for improving flee blast resistance of major varieties of rice in Jilin Prov- ince.

New technique for identifying varieties resistance to rice blast

After 8 yrs lab experiments and field tests, an advanced technique for identifying varieties resistance to rice blast was developed by a research group in Plant Protection Institute, Zhejiang Academy of Agricultural Sciences. With this technique, the inoculum was prepared on a maize-rice-straw-agar media which was suitable for sporulation of most rice blast pathogen isolates. To avoid possible virulence variation during the vegetative growth, the conidia were harvested and preserved for later different sets of resistance evaluation tests. A simple and precise new method—smear inoculation was used for infection. The experi-

Marker-Assisted Introgression of Quantitative Resistance Gene pi21 Confers Broad Spectrum Resistance to Rice Blast

The quantitative resistance gene pi21 from Sensho was introgressed to an indica breeding line IR63307-4B-13-2,a pyramiding line IRBB4/5/13/21,and a tropical japonica line Kinandang Patong by marker-assisted backcrossing.A total of 192 improved lines at the BC4F3 and BC4F4 generations were developed and confirmed to have the gene introgression via genotyping using a pi21-specific InDel marker.Thirteen randomly selected improved lines,representing all the three genetic backgrounds,demonstrated resistance against leaf blast composites in the field and a broader spectrum resistance against individual isolates compared to the recurrent parents in the glasshouse.Specifically,the tested lines exhibited pi21-acquired resistance against 11 leaf blast isolates that elicited susceptible reactions from the recurrent parents.All the tested lines maintained a comparative heading date,and similar or improved panicle length,number of primary branches per panicle and number of total grains per panicle relative to the recurrent parents.The physical grain characteristics of the recurrent parents were also maintained in the 13 lines tested,although variability in the amylose content and chalkiness degree was observed.The successful marker-assisted introgression of pi21 in diverse genetic backgrounds and the resulting broader spectrum resistance of improved lines against leaf blast indicate the potential of pi21 for deployment in cultivars grown across other rice growing regions in Asia.

Improving Rice Blast Resistance by Mining Broad-Spectrum Resistance Genes at Pik Locus

Magnaporthe oryzae is known for its genetic diversity and pathogenic variability,leading to rapid breakdown of resistance in rice.Incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity.Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles.We conducted a large-scale screen of rice blast resistance in about 2000 rice accessions.Among them,247 accessions showed at least medium resistance to the natural infection of rice blast and 7 novel Pik alleles were identified from them.Variations in gene sequences were then correlated with the phenotypic trait to enable the identification of favorable alleles.Among the seven novel Pik alleles,the resistant rate of Pik-R0/ME/7017 donors was greater than 80%,and the disease score was less than 3.Through molecular marker-assisted backcross breeding,we successfully transferred the three Pik alleles,Pik-R0/ME/7017,into an elite cultivated line Kongyu 131 to obtain BC_(3)F_(2)lines,which showed enhanced resistance to rice blast compared with the recurrent parent.Assessment of these near-isogenic lines in the greenhouse using 31 isolates of M.oryzae from Heilongjiang Province of China revealed that the resistant levels of the BC_(3)F_(2)lines with Pik-R0/ME/7017 were significantly higher than those of the established cloned resistance genes Pik-m and Pi1.Exploring such alleles will enrich our gene library for resistance to rice blast.

Development of Specific DNA Markers for Detecting the Rice Blast Resistance Gene Alleles Pi2/9/z-t

Specific molecular markers for detecting the alleles of Pi2/Pi9/Piz-t,designated as Pi2 SNP,Pi9 SNP and Pizt-PA,respectively,have been successfully developed through sequence comparison method.These three specific markers can distinctly distinguish target genes from others mapped on Pi2/9 locus,which facilitates the molecular marker-assistant selection and the development of pyramiding cultivars combined with other allele R genes.Additionally,101 main cultivars and breeding parents collected from different rice-planting areas have been diagnosed with these three specific markers,and the results showed that only 2 out of the 101 cultivars carried Piz-t,but neither Pi2 nor Pi9 band had been detected.It indicates that most of the main cultivars and breeding parents in China do not carry Pi2/Pi9/Piz-t.These results provide important information for the application of Pi2/Pi9/Piz-t in the blast resistance breeding programs.

Distribution of Eight Blast-resistant Genes in Parents of Three-line Japonica Hybrid Rice

The distribution of rice blast-resistant genes Pi2, Pi5, Pi9, Pi54, Pia, Pib, Pit and Pita in 36 restoring lines and 42 sterile lines were detected using functional molecular markers. The results indicated that those eight blast-resistant genes distributed in the 78 tested lines at various frequencies. Six rice blast-resistant genes were detected in the sterile line Shen 11 A, containing the most rice blast-resistant genes among the 78 lines, whereas no rice blast-resistant gene was detected in the lines 8527 A, Xu1 A and 016 A. The genetic distance-based clustering analysis showed that those 78 lines could be divided into five groups.

Cultivar Mixture Cooperation(CMC)of rice varieties(lines) with different blast resistant gene

We studied Cultivar Mixture Cooperation(CMC)of rice varieties(lines)by screening mixture ofrice varieties which possess different genes,1983-1996.

Analysis of the Relationship between Blast Resistance Genes and Disease Resistance of Rice Germplasm via Functional Molecular Markers

Rice blast disease is one of the most devastating diseases of rice(Oryza sativa L.)caused by the fungus Magnaporthe oryzae(M.oryzae),and neck blast is the most destructive phase of this illness.The underlying molecular mechanisms of rice blast resistance are not well known.Thus,we collected 150 rice varieties from different ecotypes in China and assessed the rice blast resistances under the natural conditions that favoured disease development in Jining,Shandong Province,China in 2017.Results showed that 92(61.3%)and 58(38.7%)rice varieties were resistant and susceptible to M.oryzae,respectively.Among the 150 rice varieties screened for the presence of 13 major blast resistance(R)genes against M.oryzae by using functional markers,147 contained one to eight R genes.The relationship between R genes and disease response was discussed by analysing the phenotype and genotype of functional markers.The results showed that the rice blast resistance gene Pita was significantly correlated with rice blast resistance.Our results provided a basis for the further understanding of the distribution of 13 major R genes of rice blast in the germplasm resources of the tested rice varieties,and were meaningful for rice disease resistance breeding.

Development of Hybrid Rice Variety FY7206 with Blast Resistance Gene Pid3 and Cold Tolerance Gene Ctb1

Hybrid rice Fanyou 7206（FY7206）, derived from the cross between a sterile line Fanyuan A and a restorer line Fuhui 7206, was bred by the Rice Research Institute, Fujian Academy of Agricultural Sciences, China. FY7206 was characterized by moderate blast resistance, cold tolerance, as well as wide adaptability, and high yields. The blast resistance results indicated that the frequencies of blast races in race B, race C and the total resistance frequency for FY7206 were 95.5%, 100.0% and 97.2%, respectively. The disease resistance results showed that the leaf blast grade for FY7206 was level 1 and panicle blast was level 5. The indoor spray results indicated that FY7206 was resistant to 11 isolates of Magnorpathe oryzae. The blast resistance of FY7206 might be derived from the high expression of blast resistance gene Pid3. The results for simulated cold resistance in an artificial climate chamber indicated that the cold tolerance for FY7206 was moderate at the booting and flowering stages. The cold tolerance results also indicated that FY7206 could be tolerant to temperatures as low as 10 °C at the seedling stage. The q RT-PCR results showed that the expression of cold tolerance gene Ctb1 in FY7206 was relatively high. These results suggested that FY7206 is a hybrid indica rice variety with good comprehensive characteristics, including blast resistance and cold tolerance.

Blast Resistance of Rice Induced by Ag-antibiotic 702

To explore rice blast resistance induced by Ag-antibiotic 702, different concentrations of Ag-antibiotic 702 were spayed on susceptible variety Luliangyou 996 at three-leaf and one-heart stage, to screen the optimal concentration for inducing rice blast resistance. Ag-antibiotic 702 was sprayed at seven different growth stages of rice, to determine the best growth stage for induced blast resistance and duration of blast resistance. Various treatments were inoculated with spore fluid of Magnaporthe grisea at 4 d post spraying, and disease index and incidence rate as well as induction effect of different treatments were investigated after 7 d. The results showed that six concentrations of Ag-antibiotic 702 could induce rice resistance against blast, and 15 μg/mL led to the highest blast resistance; spraying 15 μg/mL Ag-antibiotic 702 at seven different growth stages could induce rice resistance against blast ; the three-leaf and one-heart stage was the best growth stage for inducing rice blast resistance, and the relative induction effect reached 56.56% ; rice blast resistance was the highest at 48 -96 h post spraying, and duration of induced rice blast resistance exceeded 144 h. The study will provide useful experimental data for further development of Ag-antibiotic 702 and application of pre- vention and control methods against rice blast.

Current Status of Conventional and Molecular Interventions for Blast Resistance in Rice

Pyricularia oryzae anamorph of Magnaporthe oryzae is one of the most notorious fungal pathogens causing severe economic loss in rice production worldwide. Various methods, viz. cultural, biological and molecular approaches, are utilized to counteract this pathogen. Moreover, some tolerant or resistant rice varieties have been developed with the help of breeding programmes. Isolation and molecular characterization of different blast resistance genes now open the gate for new possibilities to elucidate the actual allelic variants of these genes via various molecular breeding and transgenic approaches. However, the behavioral pattern of this fungus breakups the resistance barriers in the resistant or tolerant rice varieties. This host-pathogen barrier will be possibly countered in future research by comparative genomics data from available genome sequence data of rice and M. oryzae for durable resistance. Present review emphasized fascinating recent updates, new molecular breeding approaches, transgenic and genomics approaches(i.e. mi RNA and genome editing) for the management of blast disease in rice. The updated information will be helpful for the durable, resistance breeding programme in rice against blast pathogen.