Diversity and genetics of nitrogen-induced susceptibility to the blast fungus in rice and wheat

Abstract

Background: Nitrogen often increases disease susceptibility, a phenomenon that can be observed under controlled conditions and called NIS, for Nitrogen-Induced Susceptibility. NIS has long been reported in the case of rice blast disease caused by the fungus Magnaporthe oryzae. We used an experimental system that does not strongly affect plant development to address the question of NIS polymorphism across rice diversity and further explored this phenomenon in wheat. We tested the two major types of resistance, namely quantitative/partial resistance and resistance driven by known resistance genes. Indeed there are conflicting reports on the effects of NIS on the first one and none on the last one. Finally, the genetics of NIS is not well documented and only few loci have been identified that may control this phenomenon.

Results: Our data indicate that NIS is a general phenomenon affecting resistance to blast fungus in these two cereals. We show that the capacity of rice to display NIS is highly polymorphic and does not correlate with difference related to indica/japonica sub-groups. We also tested the robustness of three different major resistance genes under high nitrogen. Nitrogen partially breaks down resistance triggered by the Pi1 gene. Cytological examination indicates that penetration rate is not affected by high nitrogen whereas growth of the fungus is increased inside the plant. Using the CSSL mapping population between Nipponbare and Kasalath, we identified a Kasalath locus on chromosome 1, called NIS1, which dominantly increases susceptibility under high nitrogen. We discuss the possible relationships between Nitrogen Use Efficiency (NUE), disease resistance regulation and NIS.

Conclusions: This work provides evidences that robust forms of partial resistance exist across diversity and can be easily identified with our protocol. This work also suggests that under certain environmental circumstances, complete resistance may breakdown, irrelevantly of the capacity of the fungus to mutate. These aspects should be considered while breeding for robust forms of resistance to blast disease.

Figure 1

Diversity of nitrogen-induced susceptibility in rice. Nitrogen (1 N) or no nitrogen (0 N) was added to rice plants one day before inoculation with the GUY11 isolate of M. oryzae (see Methods). A. Different types (shown in B) of lesions typical of partial resistance were counted 7 days post-inoculation (dpi): lesions typical of resistance (light grey bars) and lesions typical of susceptibility (dark grey bars). The mean of each category of lesion was compared between the 0 N and 1 N condition and statistical differences are shown by *** (p value < 0.001; Wilcoxon test). B. Examples of nitrogen-induced susceptibility at 7 dpi. Lesions typical of resistance (R) and lesions typical of susceptibility (S) are shown.

Figure 2

Robustness of resistance genes under high nitrogen treatment. Nitrogen (1 N) or no nitrogen (0 N) was added to rice plants one day before inoculation (see Methods). A. Different resistance genes (Pi1, Pi2 and Co39/Pia) were tested using different combinations of rice cultivars and M. oryzae isolates. B. The number of lesions typical of resistance was counted. There were no susceptible lesions under these conditions (white bars: 0 N; black bars: 1 N). The mean of the number of resistant lesions were compared between the 0 N and 1 N conditions and statistical differences are shown by *** (p value < 0.001; Wilcoxon test). C. At the indicated time points after inoculation, the development stage of the fungus was observed and four categories of growth stages were counted 100 interaction sites/condition). This experiment was repeated 3 times and gave similar results. D. The average size of individual fungal colonies was measured inside plant tissues. The mean and sd are shown 50 colonies counted for each condition; white bars: 0 N and black bars: 1 N). Statistical differences are shown by *** (p value < 0.001; Wilcoxon test).

Figure 3

Defense gene expression triggered by the Pi1 resistance gene under nitrogen treatment. Rice plants of the C104Lac genotype (containing the Pi1 resistance gene recognizing the avirulent isolate of M. oryzae CL367) were grown under the same conditions except one day before inoculation where they were either treated with no additional nitrogen (0 N) or additional nitrogen (1 N). Uninfected plants (white bars: Mock) were used as controls to compare to infected plants (black bars). The expression of defense-related marker genes (normalized with the actin gene; arbitrary units are given) was measured at the indicated time points after infection (2 and 4 days post inoculation-dpi). The mean and sd of three independent replicates are shown. The statistical differences between mock and inoculated plants are shown (Wilcoxon tests; *: p < 0.05; **p < 0.01; ***: p < 0.001).

Figure 4

Mapping of a locus required for resistance under high nitrogen. The Nipponbare X Kasalath CSSL mapping population was scored for NIS to the GUY11 isolate (see Methods). A. The CSSL5, CSSL19 lines and two recombinant lines (19-42-2 and 19-42-4) identify chromosome 1 as carrying a Kasalath locus conferring susceptibility under high nitrogen treatment. The portions of the chromosome in black, white and dashed respectively correspond to Kasalath, Nipponbare and heterozygous as established by microsatellites markers (RM markers indicated on the left of the figure). Possible regulators of disease resistance and nitrogen metabolism are indicated for the NIS1 locus on the right of the figure. B. Plants from the 19–42 family segregating at the NIS1 locus were grown under low (0 N) or high nitrogen (1 N) and genotyped with several markers flanking the NIS1 locus. The genotype at NIS1 is indicated. Quantitative phenotype of these group of plants with similar genotype (the number of individual plants is indicated) was established after inoculation with the GUY11 isolate. Statistical differences are shown by *** (p value < 0.001; Wilcoxon test).

Figure 5

Nitrogen-induced susceptibility to wheat blast. Nitrogen (black bars; 1 N) or no nitrogen (white bars; 0 N) was added to wheat plants one day before inoculation with the BR32 isolate of M. oryzae (see Methods). Lesions were counted 7 days post-inoculation. ***: p value < 0.001 (Wilcoxon test).