Shinzo Koizumi

Pi34-AVRPi34: a new gene-for-gene interaction for partial resistance in rice to blast caused by Magnaporthe grisea

The japonica rice (Oryza sativa) cultivar Chubu 32 has a high level of partial resistance to blast, which is mainly controlled by a dominant resistance gene located on chromosome 11. The partial resistance to the rice blast fungus (Magnaporthe grisea) in Chubu 32 has isolate specificity; isolate IBOS8-1-1 is more aggressive on Chubu 32 than are other isolates. We hypothesized that the gene-for-gene relationship fits this case of a partial resistance gene in Chubu 32 against the avirulence gene in the pathogen. The partial resistance gene in Chubu 32 was mapped between DNA markers C1172 (and three other co-segregated markers) and E2021 and was designated Pi34. In the 32 F3 lines from the cross between a chromosome segment substitution line (Pi34−) from Koshihikari/Kasalath and Chubu 32, the lines with high levels of partial resistance to the M. grisea isolate Y93-245c-2 corresponded to the presence of Pi34 estimated by graphic genotyping. This indicated that Pi34 has partial resistance to isolate Y93-245c-2 in compatible interactions. The 69 blast isolates from the F1 progeny produced by the cross between Y93-245c-2 and IBOS8-1-1 were tested for aggressiveness on Chubu 32 and rice cultivar Koshihikari (Pi34−). The progeny segregated at a 1 : 1 ratio for strong to weak aggressiveness on Chubu 32. The results suggested that Y93-245c-2 has one gene encoding avirulence to Pi34 (AVRPi34), and IBOS8-1-1 is extremely aggressive on Chubu 32 because of the absence of AVRPi34. This is the first report of a gene-for-gene relationship between a fungal disease resistance gene associated with severity of disease and pathogen aggressiveness.

Quantitative trait locus analysis of resistance to panicle blast in the rice cultivar Miyazakimochi

BackgroundRice blast is a destructive disease caused by Magnaporthe oryzae, and it has a large impact on rice production worldwide. Compared with leaf blast resistance, our understanding of panicle blast resistance is limited, with only one panicle blast resistance gene, Pb1, isolated so far. The japonica cultivar Miyazakimochi shows resistance to panicle blast, yet the genetic components accounting for this resistance remain to be determined.ResultsIn this study, we evaluated the panicle blast resistance of populations derived from a cross between Miyazakimochi and the Bikei 22 cultivar, which is susceptible to both leaf and panicle blast. The phenotypic analyses revealed no correlation between panicle blast resistance and leaf blast resistance. Quantitative trait locus (QTL) analysis of 158 recombinant inbred lines using 112 developed genome-wide and 35 previously reported polymerase chain reaction (PCR) markers revealed the presence of two QTLs conferring panicle blast resistance in Miyazakimochi: a major QTL, qPbm11, on chromosome 11; and a minor QTL, qPbm9, on chromosome 9. To clarify the contribution of these QTLs to panicle blast resistance, 24 lines homozygous for each QTL were selected from 2,818 progeny of a BC2F7 backcrossed population, and characterized for disease phenotypes. The panicle blast resistance of the lines harboring qPbm11 was very similar to the resistant donor parental cultivar Miyazakimochi, whereas the contribution of qPbm9 to the resistance was small. Genotyping of the BC2F7 individuals highlighted the overlap between the qPbm11 region and a locus of the panicle blast resistance gene, Pb1. Reverse transcriptase PCR analysis revealed that the Pb1 transcript was absent in the panicles of Miyazakimochi, demonstrating that qPbm11 is a novel genetic component of panicle blast resistance.ConclusionsThis study revealed that Miyazakimochi harbors a novel panicle blast resistance controlled mainly by the major QTL qPbm11. qPbm11 is distinct from Pb1 and could be a genetic source for breeding panicle blast resistance, and will improve understanding of the molecular basis of host resistance to panicle blast.

Incidence of thiophanate-methyl resistance in Cercospora kikuchii within a single lineage based on amplified fragment length polymorphisms in Japan

We collected 247 isolates of Cercospora kikuchii from soybean seeds with typical purple stain symptoms from 15 prefectures in Japan. Of the 247 isolates, 93 were sensitive to thiophanate-methyl, a benzimidazole used to control this soybean disease; the remaining 154 were highly resistant to the fungicide. To examine genetic variability among the population of 247 isolates, we developed amplified fragment length polymorphism (AFLP) markers. An AFLP primer pair generated DNA fingerprint polymorphisms among the sample isolates, and with the unweighted pair-grouping method to cluster arithmetic means of the similarity coefficients among all pairs of the fingerprint patterns, the isolates were divided into four lineages (I to IV). Of the 247 isolates, 225 belonged to lineage I, including all isolates that were resistant to thiophanate-methyl. To determine whether the resistance of these isolates was related to mutations in the β-tubulin gene, we amplified partial nucleotide sequences of the gene from 29 representative isolates, including 12 that were resistant to thiophanate-methyl, by means of the polymerase chain reaction. The resistant isolates had identical nucleotide sequence with a one-step change at codon 198, in which the amino acid glutamic acid had been replaced by alanine. The evidence thus suggests that thiophanate-methyl resistance might have arisen in lineage I, the largest of the four lineages.

Effects of thiophanate-methyl and azoxystrobin on the composition of Cercospora kikuchii populations with thiophanate-methyl-resistant strains

Azoxystrobin was recently registered in Japan for the control of purple seed stain of soybean caused by Cercospora kikuchii, because the pathogen has developed resistance to thiophanate-methyl. To investigate the effects of these fungicides on the frequency of C. kikuchii strains resistant to thiophanate-methyl and on the genotype structure of the population, we sowed purple-stained seeds, approximately 40% of which were infected with resistant strains, as inocula with asymptomatic seeds and applied thiophanate-methyl and azoxystrobin during the reproductive growth of soybeans. The isolation frequency of resistant strains increased more than 99% by thiophanate-methyl but was not significantly increased by azoxystrobin. In amplified fragment length polymorphism (AFLP) DNA fingerprinting, genotypic diversity was significantly decreased by thiophanate-methyl but was not affected by azoxystrobin. In addition, the similarity of the AFLP genotype structure was increased by thiophanate-methyl but not by azoxystrobin. These results suggest that thiophanate-methyl selectively inhibited the proliferation of sensitive strains, which resulted in a small number of genotypes, most of which were resistant strains. Azoxystrobin was found to nonselectively inhibit proliferation of the pathogen, which retained a large number of genotypes including thiophanate-methyl-sensitive or thiophanate-methyl-resistant strains or both.

Lesion-based analysis of leaf blast suppression in mixture of rice cultivar and a resistant near-isogenic line

Leaf blast suppression in multilines was evaluated based on the number of susceptible lesions observed in a pure stand of susceptible rice cultivar Sasanishiki, and in 1 : 1 and 1 : 3 mixtures of Sasanishiki and a resistant near-isogenic line, Sasanishiki BL4 or BL7, from 1998 to 2001. The number of lesions first observed in fields in the 1 : 1 and 1 : 3 mixtures were close to theoretical numbers calculated using the number of lesions observed in the pure stands and the ratios of the susceptible Sasanishiki in the mixtures. The ratio of the number of lesions in the 1 : 1 and 1 : 3 mixtures to the number in the pure stand was 0.29 and 0.09, respectively. The relationship between these ratios and the ratios of susceptible Sasanishiki in mixtures was defined in an equation to estimate the degree of leaf blast suppression. Validation studies for the ratios of the number of lesions in the 1 : 1 and 1 : 3 mixtures to the number in the pure stand were conducted in two different locations and showed that the ratios are almost acceptable. The calculated autoinfection to alloinfection ratio was 1.3 and 1.4 in the 1 : 1 and 1 : 3 mixtures, respectively, suggesting that the calculated ratio will affect the degree of leaf blast suppression. Thus, predictors were obtained to estimate leaf blast suppression for effective blast control in multilines.

Rice Blast Control with Sasanishiki Multilines in Miyagi Prefecture

For rice blast control with multilines, nine near-isogenic lines (NILs) with different complete blast-resistance genes were developed from a leading rice cv. Sasanishiki at Miyagi Prefectural Furukawa Agricultural Experiment Station in Japan. Four of these nine NILs, namely, the Pik, Pik-m, Piz and Piz-t lines were first registered as multilines named Sasanishiki BL (BL) in 1994, and the Pita-2, Pita and Pib lines were added to the registered NILs in 1997, 1998 and 2001, respectively. BL has been cultivated in farmers’ fields in Miyagi Prefecture from 1995, was planted on over 5,000 hectares in 1997. However, its cultivated area is now less than 800 hectares, since Sasanishiki has become unpopular. Resistance of the Sasanishiki multilines to blast has not been broken down for 7 years, and other NILs are also being developed from other leading rice cultivars in our experiment station. New rice multilines are expected to be cultivated for blast control.

Ecological and genetic studies on durable use of blast resistance in rice

Blast, caused by Pyricularia oryzae (telemorph Magnaporthe oryzae) is one of the most destructive diseases of rice. To control the disease, effective and durable use of blast resistance in rice is required because it can control the disease with less chemical application, thus reducing the environmental impact as well as the production cost. Blast resistance in rice is generally classified as complete (true resistance) or partial (field resistance). Complete resistance, controlled by a major gene, is qualitative and race-specific; however, when used by itself, it breaks down within a few years as blast races overcome the resistance. On the other hand, quantitative partial resistance is considered to be more durable than complete resistance because it is generally polygenic and not race-specific. However, partial resistance has not been fully utilized because its genetic analyses have not been insufficient and there have been few simple methods to evaluate the resistance (Koizumi 2007). For durable use of blast resistance in rice, we have been able to reduce blast in multilines and to elucidate the underlying ecological mechanisms and factors affecting the distribution of blast races. We have also identified three new blast resistance genes (one for complete resistance and two for partial resistance) and clarified the genetic background of isolate-specific variation in the partial resistance to blast. Furthermore, we have developed a simple method to evaluate partial resistance to panicle blast using cut panicles of rice. An overview of these studies is presented in this article.

Vertical distribution of leaf blast lesions in mixtures of rice cultivar Sasanishiki and its resistant near-isogenic line

The vertical distribution of leaf blast lesions caused by the fungus Pyricularia grisea was studied to estimate the degree of leaf blast suppression in rice multilines in experimental paddy fields for 4 years. Leaf blast in 1 : 1 and 1 : 3 mixtures of susceptible rice cultivar Sasanishiki and its resistant near-isogenic line, Sasanishiki BL7, developed slower than that in pure stands of Sasanishiki. The average distance of lesions on leaves from the ground in the 1 : 3 mixtures was significantly lower than that in the pure stands at the end of leaf blast epidemics (at booting stage). This result shows that the distribution of leaf blast lesions in the upper layer differs between the susceptible pure stands and the 1 : 3 mixtures at the end of leaf blast epidemics.

Spatial distribution of purple seed stain of soybean caused by Cercospora kikuchii in fields

To investigate the frequency distribution of purple seed stain of soybean caused by Cercospora kikuchii in two experimental fields in 2004, we set up rows 75 cm apart and sowed two asymptomatic seeds at each of positions 20 cm apart in each row. We sowed purple-stained seeds infected with the pathogen as inocula at four points instead of asymptomatic seeds in each field. We assessed disease incidence in harvested seeds by counting the numbers of purple-stained and asymptomatic seeds. To determine the spatial distribution of the disease, we grouped the field points into analytical units of several sizes. Beta-binomial and binomial distributions described the distribution patterns of purple-stained seeds. The smallest value of α, a beta-binomial parameter, occurred with analytical units that contained three or nine points next to each other within a single row, suggesting that these units showed the most aggregated distribution of the disease, each of the patches of seeds infected with C. kikuchii could be defined approximately by the area covered by three or nine points (75 × 60 or 75 × 180 cm), and the disease tended to infect plants next to each other within rows.