[논문]Seed-born Burkholderia glumae Infects Rice Seedling and Maintains Bacterial Population during Vegetative and Reproductive Growth Stage

Pedraza, Luz Adriana; Bautista, Jessica; Uribe-Velez, Daniel

doi:10.5423/ppj.oa.02.2018.0030

Seed-born Burkholderia glumae Infects Rice Seedling and Maintains Bacterial Population during Vegetative and Reproductive Growth Stage 원문보기

The plant pathology journal, v.34 no.5, 2018년, pp.393 - 402

Pedraza, Luz Adriana (Instituto de Biotecnologia, Universidad Nacional de Colombia) , Bautista, Jessica (Instituto de Biotecnologia, Universidad Nacional de Colombia) , Uribe-Velez, Daniel (Instituto de Biotecnologia, Universidad Nacional de Colombia)

Abstract ▼ AI-Helper

Rice world production is affected due to the growing impact of diseases such as bacterial panicle blight, produced by Burkholderia glumae. The pathogen-induced symptoms include seedling rot, grain rot and leafsheath browning in rice plants. It is currently recognized the entrance of this pathogen to the plant, from infected seeds and from environmental sources of the microorganism. However, it is still not fully elucidated the dynamics and permanence of the pathogen in the plant, from its entry until the development of disease symptoms in seedlings or panicles. In this work it was evaluated the infection of B. glumae rice plants, starting from inoculated seeds and substrates, and its subsequent monitoring after infection. Various organs of the plant during the vegetative stage and until the beginning of the reproductive stage, were evaluated. In both inoculation models, the bacteria was maintained in the plant as an endophyte between $1{\times}10^1$ and $1{\times}10^5cfu$ of B. $glumae.g^{-1}$ of plant throughout the vegetative stage. An increase of bacterial population towards initiation of the panicle was observed, and in the maturity of the grain, an endophyte population was identified in the flag leaf at $1{\times}10^6cfu$ of B. $glumae.g^{-1}$ fresh weight of rice plant, conducting towards the symptoms of bacterial panicle blight. The results found, suggest that B. glumae in rice plants developed from infected seeds or from the substrate, can colonize seedlings, establishing and maintaining a bacterial population over time, using rice plants as habitat to survive endophyticly until formation of bacterial panicle blight symptoms.

주제어

표/그림 (5)

그림 Fig. 1. Development of B. glumae disease symptoms in rice seedlings (cvF733), at 7 DAI, through two inoculum sources of the pathogen (A) Seed (vacuum inoculation) and (B) Substrate (pathogenic bacteria incorporated into the supporting substrate). Different letters show statistically significant differences between treatments, with each of the variables (stem length, root length and degree of infection), according to the Duncan test, with a level of 95.0% confidence. The graph corresponds to one of at least two repetitions in time, for both infection models, which showed the same trend and statistical differences.
표 Table 1. B. glumae population (cfu.g^-1 of fresh weight of plant) in inoculated plants by vacuumed seeds and seed/plantlet substrate, through different plant growing stages up to the reproductive phase
그림 Fig. 2. Effect of inoculation of B. glumae on stem and root length in relation to non-inoculated plants during vegetative and reproductive phases of the plant. (A) Inoculation under the vacuum seed model and (B) Inoculation in substrate (bacteria incorporated in the seed/ plantlet substrate). Different letters show statistically significant differences for each of the plant organs between the vegetative stages, according to the Duncan Test with a level of 95.0% confidence. ¶ Phases determined according to the extended BBCH scale for rice according to Hack et al. (1992) modified from Lancashire et al. (1991).
표 Table 2. Effect of B. glumae on the filling of grains and grain weight in plants inoculated from seed grown during 7 days under the substrate model
그림 Fig. 3. Agarose gel electrophoresis showing PCR products for presumptive B. glumae colonies of inoculated plants at stage of 2-4 tillers. MWM molecular weight marker. Lines: 1, control reaction without DNA sample; 2, Positive control (pure DNA sample of B. glumae 3200-12); 3-6, Presumptive colonies isolated from leaf; 7-10, Presumptive colonies isolated from stem; 11-12, Presumptive colonies isolates from root; 13, Negative control (non-presumptive colony DNA). The size of the amplicon is 286 bp.

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문제 정의

As conclusion, this study contributes to understand the population dynamic and distribution of B. glumae in different plant tissues, starting from artificially infected seeds or plant substrates. It was determined that once the bacterium enters the plant, it can mobilize through different organs and produce disease symptoms during the seedling phase; can be maintained as endophyte in the rice plant within the vegetative growth, even without showing very evident disease symptoms.
Besides, it is not even clear what happens to bacteria during vegetative growth of the plant after that kind of infection, because those studies do not fully solve the question of whether it remains on the ground, epiphytically or as endophyte in the plant, particularly when rice seeds have been inoculated (Hickichi, 1993). In this context, the aim of this study was to determine the colonization capacity of B. glumae during vegetative and reproductive stage of the rice plant, starting from seed infection, both from asymptomatic seeds, carrying a known population of the bacteria and from B. glumae free rice seeds, planted on water agar as substrate with a known concentration of B. glumae.

제안 방법

PCR products were evaluated on 1% agarose gels according to the method of Sambrook and Rusell (2001). Finally, some of the PCR products during the evaluations of the different inoculation models were randomly selected from each of the evaluation times, for sequencing them by the method of Sanger at the Institute of Genetics of the Universidad Nacional de Colombia, to confirm the sequences identity. In order to monitor B.
For both models, the B. glumae inoculum was prepared as described above, and adjusted to concentrations, between 1.0 × 105 - 1.0 × 108 cfu.ml-1 depending on the assay, to determine the most adequate concentration for the development of disease symptoms in rice seedlings.
In both cases, the stem and root length of the seedlings were measured and assigned a level within the disease severity scale proposed by Flórez-Zapata and Uribe-Vélez (2011). For the tests, a Petri dish with five rice seeds and three replicates per treatment were used as experimental unit, repeating three times in time to confirm the reproducibility of the results. In the case of the seed inoculation model, disinfected seeds not inoculated with the bacteria and subjected to the same vacuum conditions, were used as negative control; for the substrate inoculation, disinfected seeds were used on a water agar without bacterial inoculum.
For this reason, in this work, we developed a model of infection, using the substrate supporting the plant, with up to 1 × 107 cfu of B. glumae.ml-1 of substrate, as carrier of the pathogen.
Surface sterilization of seeds was tested by putting seeds on nutrient agar (Oxoid® UK) and King B (gl-1: Protease peptone, 20; KH2PO4, 1.5; MgSO4.7H2O, 1.5, anhydrous glycerol, 8.7 ml, bacteriological agar, 14.0), to evaluate persistence of external contaminants.
determine if B. glumae infected seedlings, are maintained as endophytes throughout the life cycle of the plant, until grain formation, a similar experiment as described in previous section, was done using a growth chamber (Sanyo®, Japan) at 28°C ± 1°C, 4650 ± 100 lux, relative humidity of 75% and a photoperiod of 12:12 light:darkness using different sampling days.

대상 데이터

Plants were incubated until processing, according to different vegetative stages selected for a total of four evaluation times, the first was at 10 days of inoculation with 1 or 2 unfolded leaves, for the second stage the seedlings had 3 unfolded leaves, for the third stage they already had 7 to 8 unfolded leaves and for the last stage the plants had 9 or more unfolded leaves. For each day of evaluation, three replicates using one plant per replicate was sampled, except for the first evaluation in which 20 seedlings were taken per replicate. At all times of evaluation, stem and root length were determined.

데이터처리

The Shapiro-Wilk Test was used with 95% significance to determine the normality of the data and the Levene Test with 95% confidence to determine the homoscedasticity of the data. In the case of having parametric data, the ANOVA table was used for the analysis of variance homogeneity and in the case of showing differences between treatments, a Duncan multiple comparison test was performed with a 95% confidence interval. When non-parametric data were obtained, data transformation was used according to the kurtosis of the distribution of the data and when this was not possible, the Kuskall-Wallis test was used.

이론/모형

In the case of having parametric data, the ANOVA table was used for the analysis of variance homogeneity and in the case of showing differences between treatments, a Duncan multiple comparison test was performed with a 95% confidence interval. When non-parametric data were obtained, data transformation was used according to the kurtosis of the distribution of the data and when this was not possible, the Kuskall-Wallis test was used.

성능/효과

3). An important percentage of PCR products obtained, were confirmed by sequencing, using Sanger technique, in order to determine whether they actually corresponded to the genome of B. glumae CIAT3200-12, finding in all cases a 100% identity. This confirmation was carried out on all evaluated days as well as for all forms of inoculation.
Bacterial infection of rice seedlings by vacuum infected seeds, using different concentrations of B. glumae, showed the development of disease symptoms in 80 to 93% of inoculated plants. The infection showed a dose response effect within evaluated concentrations (1.
glumae in different plant tissues, starting from artificially infected seeds or plant substrates. It was determined that once the bacterium enters the plant, it can mobilize through different organs and produce disease symptoms during the seedling phase; can be maintained as endophyte in the rice plant within the vegetative growth, even without showing very evident disease symptoms. However, bacterial concentration internally increases subtly when the plant enters the reproductive stage, being more remarkable in the flag leaf, ending with panicle blight symptoms, with production of significant number of empty grains.
glumae disease symptoms. It was found, that non-inoculated plants showed a significant lower percentage of empty grains (25.72%), in relation to plants inoculated with B. glumae, that showed 58.0% empty grains (Table 2). In the same sense, the weight of 100 grains adjusted to 14% moisture, was higher in control plants with a value of 7.
1B). Stem and root length variables showed also a reduction in size, with statistically significant differences for all the concentrations of B. glumae, in comparison with un-inoculated control seedlings. However, no differences were found between them, except for the stem length at the concentration 1.
g^-1 of leaf) (Table 1). These results show that at the beginning of the reproductive phase, the bacterial population increased towards the aerial organs of the plant, especially towards the stem and leaf pod, and decreased in the root. Plants at the stage of grain ripening showed that bacterial population maintained the size at the root (6.
2A, B). These results showed again, that stem length was an agronomic variable affected by the inoculation of B. glumae in most stages of the vegetative phase and at the beginning of the reproductive phase, independently to the source of inoculum.

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표제어: PCR

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