A Rapid Survey of Avirulence Genes in Field Isolates of Magnaporthe oryzae

Magnaporthe oryzae is the causal agent for the devastating disease rice blast. The avirulence (AVR) genes in M. oryzae are required to initiate robust disease resistance mediated by the corresponding resistance (R) genes in rice. Therefore, monitoring pathogen AVR genes is important to predict the stability of R gene-mediated blast resistance. In the present study, we analyzed the DNA sequence dynamics of five AVR genes, namely, AVR-Pita1, AVR-Pik, AVR-Pizt, AVR-Pia, and AVR-Pii, in field isolates of M. oryzae in order to understand the effectiveness of the R genes, Pi-ta, Pi-k, Pi-zt, Pia, and Pii in the Southern U.S. rice growing region. Genomic DNA of 258 blast isolates collected from commercial fields of the Southern UNITED STATES during 1975–2009 were subjected to PCR amplification with AVR gene-specific PCR markers. PCR products were obtained from 232 isolates. The absence of PCR products in the remaining 26 isolates suggests that these isolates do not contain the tested AVR genes. Amplified PCR products were subsequently gel purified and sequenced. Based on the presence or absence of the five AVR genes, 232 field isolates were classified into 10 haplotype groups. The results revealed that 174 isolates of M. oryzae carried AVR-Pita1, 225 isolates carried AVR-Pizt, 44 isolates carried AVR-Pik, 3 isolates carried AVR-Pia, and one isolate carried AVR-Pii. AVR-Pita1 was highly variable, and 40 AVR-Pita1 haplotypes were identified in avirulent isolates. AVR-Pik had four nucleotide sequence site changes resulting in amino acid substitutions, whereas three other AVR genes, AVR-Pizt, AVR-Pia, and AVR-Pii, were relatively stable. Two AVR genes, AVR-Pik and AVR-Pizt, were found to exist in relatively larger proportions of the tested field isolates, which suggested that their corresponding R genes Pi-k and Pi-zt can be deployed in preventing blast disease in the Southern UNITED STATES in addition to Pi-ta. This study demonstrates that continued AVR gene monitoring in the pathogen population is critical for ensuring the effectiveness of deployed blast R genes in commercial rice fields.

Download Full-text

Novel haplotypes and networks of AVR-Pik alleles in Magnaporthe oryzae

10.1101/499335 ◽

2018 ◽

Author(s):

Jin-bin Li ◽

Qun Wang ◽

Chengyun Li ◽

Yunqing Bi ◽

Xue Fu ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Yunnan Province ◽

Sequence Variation ◽

Specific Resistance ◽

Blast Disease ◽

Base Substitution ◽

R Genes ◽

Dna Sequence Variation ◽

Pcr Products ◽

Avr Genes

Rice blast disease is one of the most destructive fungal diseases of rice world-wide. The avirulence (AVR) genes of Magnaporthe oryzae are recognized by the cognate resistance (R) genes of rice, and trigger race specific resistance. Here, we studied the possible evolutionary pathways in the evolution of AVR-Pik alleles by analyzing the DNA sequence variation and assayed for their avirulence function to the cognate Pik alleles resistance genes under field conditions in China. Results of PCR products showed that 278 isolates of M. oryzae carry AVR-Pik alleles among genomic DNA of 366 isolates of M. oryzae collected from Yunnan Province, China. Among of them, 66.7-90.3% of M. oryzae carry AVR-Pik alleles from six regions of Yunnan. Moreover, 10 AVR-Pik haplotypes encoding five novel AVR-Pik variants were identified among 201 isolates. The AVR-Pik alleles stepwise evolved to virulence from avirulent forms via base substitution. These findings demonstrate that AVR-Pik alleles are under positive selection and mutations are responsible for defeating race-specific resistance Pik alleles in nature.

Download Full-text

Avirulence (AVR) Gene-Based Diagnosis Complements Existing Pathogen Surveillance Tools for Effective Deployment of Resistance (R) Genes Against Rice Blast Disease

Phytopathology ◽

10.1094/phyto-12-16-0451-r ◽

2017 ◽

Vol 107 (6) ◽

pp. 711-720 ◽

Cited By ~ 13

Author(s):

S. M. Selisana ◽

M. J. Yanoria ◽

B. Quime ◽

C. Chaipanya ◽

G. Lu ◽

...

Keyword(s):

Rice Blast ◽

Polymerase Chain Reaction Amplification ◽

Rice Blast Disease ◽

Blast Disease ◽

R Gene ◽

R Genes ◽

Pathogen Population ◽

Avr Gene ◽

Avr Genes ◽

Virulence Spectrum

Avirulence (AVR) genes in Magnaporthe oryzae, the fungal pathogen that causes the devastating rice blast disease, have been documented to be major targets subject to mutations to avoid recognition by resistance (R) genes. In this study, an AVR-gene-based diagnosis tool for determining the virulence spectrum of a rice blast pathogen population was developed and validated. A set of 77 single-spore field isolates was subjected to pathotype analysis using differential lines, each containing a single R gene, and classified into 20 virulent pathotypes, except for 4 isolates that lost pathogenicity. In all, 10 differential lines showed low frequency (<24%) of resistance whereas 8 lines showed a high frequency (>95%), inferring the effectiveness of R genes present in the respective differential lines. In addition, the haplotypes of seven AVR genes were determined by polymerase chain reaction amplification and sequencing, if applicable. The calculated frequency of different AVR genes displayed significant variations in the population. AVRPiz-t and AVR-Pii were detected in 100 and 84.9% of the isolates, respectively. Five AVR genes such as AVR-Pik-D (20.5%) and AVR-Pik-E (1.4%), AVRPiz-t (2.7%), AVR-Pita (0%), AVR-Pia (0%), and AVR1-CO39 (0%) displayed low or even zero frequency. The frequency of AVR genes correlated almost perfectly with the resistance frequency of the cognate R genes in differential lines, except for International Rice Research Institute-bred blast-resistant lines IRBLzt-T, IRBLta-K1, and IRBLkp-K60. Both genetic analysis and molecular marker validation revealed an additional R gene, most likely Pi19 or its allele, in these three differential lines. This can explain the spuriously higher resistance frequency of each target R gene based on conventional pathotyping. This study demonstrates that AVR-gene-based diagnosis provides a precise, R-gene-specific, and differential line-free assessment method that can be used for determining the virulence spectrum of a rice blast pathogen population and for predicting the effectiveness of target R genes in rice varieties.

Download Full-text

Identification of Magnaporthe oryzae Avirulence Genes to Three Rice Blast Resistance Genes

Plant Disease ◽

10.1094/pdis.2004.88.3.265 ◽

2004 ◽

Vol 88 (3) ◽

pp. 265-270 ◽

Cited By ~ 13

Author(s):

C. X. Luo ◽

Y. Fujita ◽

N. Yasuda ◽

K. Hirayae ◽

T. Nakajima ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Goodness Of Fit ◽

Blast Resistance ◽

Random Amplified Polymorphic Dna ◽

Rice Cultivars ◽

Avirulence Genes ◽

Rice Blast Resistance ◽

Parental Isolate ◽

Avr Gene ◽

Avr Genes

The segregation of avirulence/virulence was studied in 115 F1 progeny isolates of Magnaporthe oryzae from a cross of two field isolates on three Japanese race-differential rice cultivars Kanto 51, Fukunishiki, and Toride 1. The χ2 tests of goodness-of-fit for a 1:1 ratio indicated that avirulence on cvs. Kanto 51, Fukunishiki, and Toride 1 was under monogenic control. The relationship between the avirulence (Avr) gene in the parental isolate and the Avr gene in the standard isolate was investigated by using 100 lines each of three F3 families from the crosses of the rice cultivars Norin 3/Kanto 51, AK61/Fukunishiki, and Norin 3/Toride 1, respectively. Based on the resistant reactions of the F3 rice lines to the parental isolates and the standard isolates harboring three known Avr genes, three genetically independent Avr genes, AvrPik, AvrPiz, and AvrPiz-t, were identified. The three identified Avr genes were mapped using random amplified polymorphic DNA (RAPD) analysis, and a partial linkage map was constructed with 17 RAPD markers closely linked to the Avr genes. Twelve markers and AvrPik, three markers and AvrPiz, and two markers and AvrPiz-t, as well as mating locus MAT1, constructed linkage groups A, B, and C, respectively.

Download Full-text

Osa-miR162a balances rice yield and resistance to Magnaporthe oryzae

10.21203/rs.2.22473/v1 ◽

2020 ◽

Author(s):

Xu-Pu Li ◽

Xiao-Chun Ma ◽

He Wang ◽

Yong Zhu ◽

Xin-Xian Liu ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Rice Blast ◽

Rice Yield ◽

Blast Resistance ◽

Blast Disease ◽

Mirna Biogenesis ◽

Trade Off ◽

Rice Blast Resistance ◽

Defense Related Gene ◽

Transgenic Lines

Abstract MicroRNAs (miRNAs) play essential roles in rice immunity against Magnaporthe oryzae, the causative agent of rice blast disease. Osa-miR162a targets Dicer-like 1 (DCL1) genes, which play vital roles in miRNA biogenesis and act as negative regulators in rice immunity. Here we demonstrate that Osa-miR162a improves rice immunity against M. oryzae and balances the trade-off between rice yield and resistance. Overexpression of Osa-miR162a compromises rice susceptibility to M. oryzae accompanying enhanced induction of defense-related genes and accumulation of hydrogen peroxide (H2O2). In contrast, blocking miR162 by overexpressing a target mimic of miR162 enhances susceptibility to blast fungus associating with compromised induction of defense-related gene expression and H2O2 accumulation. Moreover, the transgenic lines overexpressing Osa-miR162a display decreased seed setting rate resulting in reduced yield per plant, whereas blocking miR162 leads to an increased number of grains per panicle, resulting in increased yield per plant. Altered accumulation of miR162 had limited impact on the expression of OsDCL1. Together, our results indicate that Osa-miR162a improves rice blast resistance and plays a role in the balance of trade-off between resistance and yield.

Download Full-text

Exploring the Distribution of Blast Resistance Alleles at the Pi2/9 Locus in Major Rice-Producing Areas of China by a Novel Indel Marker

Plant Disease ◽

10.1094/pdis-10-19-2187-re ◽

2020 ◽

Vol 104 (7) ◽

pp. 1932-1938

Author(s):

Dagang Tian ◽

Yan Lin ◽

Ziqiang Chen ◽

Zaijie Chen ◽

Fang Yang ◽

...

Keyword(s):

Blast Resistance ◽

Resistance Breeding ◽

Field Trials ◽

Indel Marker ◽

Blast Disease ◽

Cereal Crops ◽

R Genes ◽

Rice Varieties ◽

Environment Friendly ◽

The World

Rice blast disease caused by the fungus Magnaporthe oryzae damages cereal crops and poses a high risk to rice production around the world. Currently, planting cultivars with resistance (R) genes is still the most environment-friendly approach to control this disease. Effective identification of R genes existing in diverse rice cultivars is important for understanding the distribution of R genes and predicting their contribution to resistance against blast isolates in regional breeding. Here, we developed a new insertion/deletion (InDel) marker, Pigm/2/9InDel, that can differentiate the cloned R genes (Pigm, Pi9, and Pi2/Piz-t) at the Pi2/9 locus. Pigm/2/9InDel combined with the marker Pi2-LRR for Pi2 was applied to determine the distribution of these four R genes among 905 rice varieties, most of which were collected from the major rice-producing regions in China. In brief, nine Pigm-containing varieties from Fujian and Guangdong provinces were identified. All of the 62 Pi2-containing varieties were collected from Guangdong, and 60 varieties containing Piz-t were from seven provinces. However, Pi9 was not found in any of the Chinese varieties. The newly identified varieties carrying the Pi2/9 alleles were further subjected to inoculation tests with regional blast isolates and field trials. Our results indicate that Pigm and Pi2 alleles have been introgressed for blast resistance breeding mainly in the Fujian and Guangdong region, and Pi9 is a valuable blast resistance resource to be introduced into China.

Download Full-text

Genotyping-by-Sequencing-Based Genetic Analysis of African Rice Cultivars and Association Mapping of Blast Resistance Genes Against Magnaporthe oryzae Populations in Africa

Phytopathology ◽

10.1094/phyto-12-16-0421-r ◽

2017 ◽

Vol 107 (9) ◽

pp. 1039-1046 ◽

Cited By ~ 4

Author(s):

Emmanuel M. Mgonja ◽

Chan Ho Park ◽

Houxiang Kang ◽

Elias G. Balimponya ◽

Stephen Opiyo ◽

...

Keyword(s):

Genetic Diversity ◽

Association Mapping ◽

Magnaporthe Oryzae ◽

Rice Blast ◽

Blast Resistance ◽

Genotyping By Sequencing ◽

Rice Breeding ◽

Rice Cultivars ◽

R Genes ◽

African Rice

Understanding the genetic diversity of rice germplasm is important for the sustainable use of genetic materials in rice breeding and production. Africa is rich in rice genetic resources that can be utilized to boost rice productivity on the continent. A major constraint to rice production in Africa is rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae. In this report, we present the results of a genotyping-by-sequencing (GBS)-based diversity analysis of 190 African rice cultivars and an association mapping of blast resistance (R) genes and quantitative trait loci (QTLs). The 190 African cultivars were clustered into three groups based on the 184K single nucleotide polymorphisms generated by GBS. We inoculated the rice cultivars with six African M. oryzae isolates. Association mapping identified 25 genomic regions associated with blast resistance (RABRs) in the rice genome. Moreover, PCR analysis indicated that RABR_23 is associated with the Pi-ta gene on chromosome 12. Our study demonstrates that the combination of GBS-based genetic diversity population analysis and association mapping is effective in identifying rice blast R genes/QTLs that contribute to resistance against African populations of M. oryzae. The identified markers linked to the RABRs and 14 highly resistant cultivars in this study will be useful for rice breeding in Africa.

Download Full-text

USE OF RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TO IDENTIFY SHORT-DAY GRANO-TYPE ONION CULTIVARS

HortScience ◽

10.21273/hortsci.28.4.271b ◽

1993 ◽

Vol 28 (4) ◽

pp. 271B-271

Author(s):

Virginia P. Roxas ◽

Ellen B. Peffley

Keyword(s):

United States ◽

Allium Cepa ◽

Random Amplified Polymorphic Dna ◽

Southern United States ◽

Random Primers ◽

Short Day ◽

Pcr Products

Nineteen random primers yielded 36 PCR-amplified products of Allium cepa profiles of each of 15 short-day grano-type onionsgrown commercially in Texas and Southern United States were compared. Several PCR productswere unique among the cultivars and can be used to differentiate among the onion cultivars investigated. A phenogram of the cultivars based on the co-occurrences of the PCR products was derived.

Download Full-text

Characterization of Molecular Identity and Pathogenicity of Rice Blast Fungus in Hunan Province of China

Plant Disease ◽

10.1094/pdis-03-16-0288-re ◽

2017 ◽

Vol 101 (4) ◽

pp. 557-561 ◽

Cited By ~ 7

Author(s):

Junjie Xing ◽

Yulin Jia ◽

Zhirong Peng ◽

Yinfeng Shi ◽

Qiang He ◽

...

Keyword(s):

Rice Blast ◽

Blast Resistance ◽

Pcr Amplification ◽

Blast Disease ◽

Hunan Province ◽

R Genes ◽

Rice Blast Resistance ◽

Molecular Identity ◽

Polymerase Chain

The blast (Magnaporthe oryzae) resistance (R) gene is the most economical and environmental method to control rice blast disease. Characterization of molecular identity and pathogenicity of M. oryzae benefits the deployment of effective blast R genes. In order to identify blast R genes that would be effective in Hunan Province,182 M. oryzae strains were analyzed with a Chinese differential system (CDS), repetitive element-based polymerase chain reaction (rep-PCR), and the presence and absence of avirulence (AVR) genes by PCR amplification with gene-specific primers. Identified blast R genes were validated with 24 monogenic lines (ML) carrying 24 major R genes. In total, 28 races (isolates) of M. oryzae was identified with CDS, and classified into 20 distinct groups with rep-PCR. Interestingly, AVR-Pia, AVR-Pik, AVR-Pizt, AVR-Pib, and AVR-Pi9 were detected in more than 86.8% of the isolates; AVR-Pita1 was in 51.3% and AVR-Pii was in only 2.5%. In contrast, pathogenicity assays on 24 ML demonstrated that Pi9, Piz5, Pikh, and Pikm were more effective, with resistant frequencies of 91.6, 91, 87.9, and 87.3%, respectively; Pia, Piks, Pit, Pi12, and Pib were less than 15%. These findings revealed the complexity of a genetic basis of rice blast resistance, and shed light on useful blast R genes in Hunan Province.

Download Full-text

Frequencies and Variations of Magnaporthe oryzae Avirulence Genes in Hunan Province, China

Plant Disease ◽

10.1094/pdis-01-21-0008-re ◽

2021 ◽

Author(s):

Zhirong Peng ◽

Ling Li ◽

Shenghai Wu ◽

Xiaolin Chen ◽

Yinfeng Shi ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Blast Resistance ◽

Hunan Province ◽

Start Codon ◽

Nucleotide Polymorphisms ◽

Avirulence Genes ◽

Rice Varieties ◽

Synonymous Mutations ◽

Pathogen Populations ◽

Avr Genes

Rice blast caused by Magnaporthe oryzae poses significant threaten to rice production. For breeding and deploying resistant rice varieties, it is essential to understand the frequencies and genetic variations of avirulence (AVR) genes in the pathogen populations. In this study, 444 isolates were collected from Hunan Province, China in 2012, 2015, and 2016, and their pathogenicity was evaluated by testing them on monogenic rice lines carrying resistance (R) genes Pita, Pizt, Pikm, Pib, or Pi9. The frequencies of corresponding AVR genes AVRPizt, AVRPikm, AVRPib, AVRPi9, and AVRPita were characterized by amplification and sequencing these genes in the isolates. Both Pi9 and Pikm conferred resistance to over 75% of the tested isolates, while Pizt, Pita, and Pib were effective against 55.63%, 15.31%, and 3.15% of the isolates, respectively. AVRPikm and AVRPi9 were detected in 90% of the isolates and AVRPita, AVRPizt, and AVRPib were present in 26.12%, 66.22%, and 79% of the isolates, respectively. Sequencing of AVR genes showed that most mutations were single nucleotide polymorphisms (SNPs), transposon insertions, and insertion mutations. The variable sites of AVRPikm and AVRPita were mainly located in the coding sequence (CDS) regions, and most were synonymous mutations. A 494 bp Pot2 transposon sequence insertion was found at the 87 bp upstream of the start codon in AVRPib. Noteworthy, although no mutations were found in CDS of AVRPi9, a GC-rich inserted sequence of ~200 bp was found at the 1272 bp upstream of the start codon in three virulent isolates. As AVRPikm and AVRPi9 were widely distributed with low genetic variation in the pathogen population, Pikm and Pi9 should be promising genes for breeding rice cultivars with blast resistance in Hunan.

Download Full-text