Screening of Arabidopsis accessions for GP17-2-mediated ISR with regard to the percent infected leaves per plant
Five days after challenge with Pst pathogen, the plants developed typical bacterial speck disease symptoms, consisting of necrotic or water-soaked spots surrounded by extensive chlorosis. Induced protection against the pathogen was quantified by determining the percent infected leaves per plant. Our results indicated a wide variation among the Arabidopsis accessions for percent infected leaves both as basal and induced resistance marker. As shown in Figure 1, percent infected leaves in control-treated plants varied over a range of 30–100% in the different accessions, where the lowest percentage of infected leaves was observed in Ra-0 and the highest was found in several accessions including Col-0. Similarly, accessions were differentially responsive to GP17-2 treatment in reducing the number of Pst-infected leaves compared to non-treated plants. Among the 75 accessions tested, 49 showed (65%) a significant reduction in percent-infected leaves per plants. The percent-infected leaves in GP17-2-treated plant ranged from 24 to 99%, where accession with the fewest percent infected leaves was En-0 and accession exhibiting greatest number of infected leaves was Cvi-0. In GP17-2-inducible ecotypes, the reduction in percent infected leaves was recorded to be 25–55% and was consistent in three independent experiments. The greatest reduction in percent infected leaves due to GP17-2 treatment was recorded in Bla-3 (55%) and the lowest was in Bu-0 (Figure 2).
Screening of Arabidopsis accessions for GP17-2-mediated ISR with regard to the percent disease severity per plant
Results in Figure 3 show that percentage area of leaf with symptoms by the disease lesions (disease severity) varied across the non-treated and GP17-2-treated Arabidopsis accessions screened. The disease severity amongst control-treated accessions ranged from 12 to 100%. There was a high severity of the disease on several accessions including Col-0, Be-1, Bla-1, Bla-3, Blh-1, Bu-17, Col-gl, Cvi-0, Pn-0, Uk-3, Van-0 and Xxx-0 accession. The accessions showing the lowest disease severity was Dra-2. The severity of the disease symptoms in many of these accessions were greatly reduced by GP17-2 treatment. Our results indicated that Arabidopsis accessions treated with GP17-2 were generally more resistant to foliar diseases caused by Pst and the disease severity in ISR-expressing and non-expressing plants ranged from 12 to 44%. However, a total of 49 (65%) GP17-2-treated accessions exhibited a significant reduction in disease severity compared with non-induced control plants, showing a 29–66% lower proportion of leaf surface with disease symptoms. Among these accessions, Bla-1 expressed the highest reduction in disease severity, while Jl-5 was the accession that exhibited the lowest reduction after GP17-2 treatment (Figure 2).
Screening of Arabidopsis accessions for GP17-2-mediated ISR with regard to the foliar population of Pst
Determination of the number of colony-forming units (cfu) of Pst in challenged leaves revealed that size of foliar population of Pst likewise varied among accessions both in control and GP17-2 treated plants. In control-treated accessions, Pst population ranged between 1.53 × 107 and 56.43 × 107 cfu/g leaves, where the highest Pst population was found in Pn-0, while Ba-1 exhibited the lowest (Figure 4). However, proliferation of Pst was significantly inhibited due to GP17-2 treatment in the same 49 accessions that showed significant reduction in infected leaf number and disease severity. Compared with control-treated plants of these accessions, GP17-2-treated plants showed a 53–84% decrease in growth of Pst in challenged leaves, demonstrating that level of suppression was more pronounced for pathogen proliferation than for number of infected leaves and percent disease severity (Figure 2). Accession with maximum decrease in growth of Pst was Ler-2, while Oy-0 exhibited the lowest reduction. Clear differences in the inhibition of bacterial proliferation in leaves of GP17-2-treated plants indicate that symptom reduction is associated with inhibition of bacterial proliferation (Figure 4).
Correlation between disease incidence or symptom severity and bacterial population
Analysis was made to determine how far expression of GP17-2-mediated ISR depends on the level of basal resistance in Arabidopsis ecotypes to Pst. Percent infected leaves and symptom severity due to Pst infection are equivalent phenotypic indicators of host basal resistance to the bacterium. When a large number of accessions is used, the estimation of infected leaf number will considerably save time compared to that of disease severity. However, the variation in basal resistance with respect to disease severity was much wide ranging than to number of infected leaves. Therefore, disease severity provides more appropriate phenotype of basal resistance in ecotypes to Pst infection. Based on the mean disease severity in control-treated plants, individual accession was hypothetically categorized as either highly susceptible, moderately susceptible (70–40%) or lowly susceptible (<40%). Infection phenotypes with disease severity >70% were classified as highly susceptible and those with disease severity of 70–40% and <40% were classified as moderately and lowly susceptible, respectively (Table 2). Out of 75 accessions evaluated, 25 accessions were found to fall within highly susceptible group. Of the remaining accessions, 23 accessions were moderately susceptible and 27 were lowly susceptible. Among the 25 highly susceptible accessions, 24 (96%) showed significant level of GP17-2-mediated ISR against Pst, when disease severity was compared between GP17-2-treated and non-treated plants (Table 2). Similarly, out of 23 moderately susceptible accessions group, 20 (86.95%) showed significantly reduced disease severity in GP17-2-treated plants compared to non-treated plant (Table 2). On the other hand, only 5 (18.5%) of 27 lowly susceptible accessions were responsive to GP17-2-treatment in showing significant level of ISR against the bacterium (Table 2). These indicate that most plants have higher expression of GP17-2-mediated ISR despite higher degrees of infection. The Chi Square (χ2 = 10.36; df = 2; 0.001 < P<0.010) test revealed a significant difference in ISR-responsiveness among different Pst susceptibility accession groups in Arabidopsis. In other words, ISR-responsiveness was not equally distributed across the different levels of host susceptibility in Arabidopsis accessions towards Pst. Accessions that had higher Pst susceptibility were likely to be more ISR-responsive than accessions that had less susceptibility. In order to elucidate a clear functional relationship between the host basal resistance and the GP17-2-induced ISR responsiveness of Arabidopsis ecotypes, we calculated the correlation between the symptom severity in control-treated ecotypes and the reduction in symptom severity in GP17-2-treated plants. There was a significant positive relationship observed between the symptom severity in control-treated ecotypes and the reduction in symptom severity due to GP17-2 treatment in the same plants, r (223) = 0.84, P < 0.000. That means percent reduction in symptom severity in ISR-inducible ecotypes linearly increased as percent disease severity increased in control-treated plants. Among all accessions, 70% of the values fit the model (Figure 5). These results indicate the ISR-inducible phenotype of Arabidopsis ecotypes was highly correlated with a relatively low level of basal resistance against Pst.