Copyright © 2020 Japanese Society for Plant Biotechnology

Plant Biotechnology 37, 343–347 (2020)DOI: 10.5511/plantbiotechnology.20.0210a

A method for evaluating root-knot nematode infection in rice using a transparent paper pouch

Hidehiko Sunohara1, Shingo Kaida2, Shinichiro Sawa1,*1 Faculty of Advanced Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan; 2 Faculty of Science, Kumamoto University, Kumamoto 860-8555, Japan* E-mail: sawa@kumamoto-u.ac.jp Tel & Fax: +81-96-342-3439

Received January 7, 2020; accepted February 10, 2020 (Edited by E. Ito)

Abstract The root-knot nematode (RKN) Meloidogyne incognita is one of the most economically damaging plant-parasitic nematodes. Molecular studies of the plant–RKN interaction have been vigorously carried out in dicotyledonous model plants, while the host range of M. incognita is wide including monocotyledonous plants. As M. incognita causes quality and yield losses in rice (Oryza sativa L.) cultivated in both upland and irrigated systems, we developed a method to examine the plant–RKN interaction in this model monocotyledonous crop plant. Here, we show that a transparent paper pouch could be used to evaluate nematode infection rates in rice with similar results to that of the traditional soil method. The system using a transparent paper pouch can be used to observe the spatial and temporal distribution of developing galls and can save the space of growth chamber.

Key words: M. incognita, paper pouch, protocol, rice.

Root-knot nematodes (RKNs, Meloidogyne spp.) are one of the most economically damaging genera of plant-parasitic nematodes affecting horticultural and field crops worldwide. RKNs are soil-borne plant pathogens that infect a wide range of plant species, including many major crop plants such as coffee (Coffea sp.), cotton (Gossypium sp.), maize (Zea mays), and rice (Oryza sativa L.). After hatching from eggs in the soil, these parasites enter root tips intercellularly and migrate to the region of cell elongation. In this region, RKNs produce multinucleate giant cells, from which they obtain water and nutrients (Bartlem et al. 2014; Favery et al. 2016). RKN-infected roots produce gall-like organs and disrupt the physiology of the root, reducing crop yield and quality.

Among the major tropical species of Meloidogyne is the southern root-knot nematode M. incognita. This RKN has an extensive host range, including many crops, with global distribution (Mai 1985; Moens et al. 2009; Sasser and Freckman 1987), and is responsible for tremendous losses in rice yield (Claudius-Cole et al. 2018). Knowledge of crop pathogens, including plant-parasitic nematodes, can be used to mitigate the risks of damage and yield loss (De Waele and Elsen 2007). In addition to its economic value as a staple food crop for more than half of the world’s population, rice is an important monocotyledonous model plant (Nguyễn et al. 2014). Many researchers use rice plants growing in soil

to evaluate the rice–nematode interaction for genetic studies (Dimkpa et al. 2016; Galeng-Lawilao et al. 2018; Lawilao et al. 2019). However, the growing process of gall in soil cannot be observed over time.

Here, we developed a new method that enabled us to observe the developing galls in the whole root using a paper pouch system. Nematode infection rates were similar in plants grown in soil and those grown in our paper pouch system.

The M. incognita strain used in this study was isolated from Koshi (Kumamoto, Japan) and was cultivated and used for infection assays as described by Nishiyama et al. (2015). And the 14 rice (Oryza sativa L.) varieties used in this study were distributed by the National Agriculture and Food Research Organization (NARO) Genebank and Nagoya University, Japan. Taichung 65 (T65) and Nipponbare were used as positive controls and are susceptible to M. incognita infection (Nguyễn et al. 2014).

We developed a new system to study the rice–nematode interaction using a transparent paper pouch and compared the nematode infection rate in this system with that of plants grown in soil.

The evaluation system using soil is as follows. Rice seeds were soaked in sterilizing solution for reduce the contamination of mold and bacteria, 1000 times dilution of commercial bleach for kitchen (Kao Corporation), for 3 days at 26°C to induce germination, and then sown on granulated soil (Kenbyo, YAE NOGEI CO., LTD.) using

This article can be found at http://www.jspcmb.jp/Published online July 30, 2020

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a cell tray system (49×49×56.5 mm/cell: FP CHUPA CORPORATION). One seed was sown per cell. The germinated seeds on soil were placed at 26°C in darkness for 3 days. The seeds were then placed in 12 h light at 26°C and 12 h dark at 24°C for 8 days. Fourteen days after the soaking period, each plant was inoculated with 800 M. incognita individuals at the J2 growth stage by adding 2 ml of a 400 individuals/ml solution to the surface of the soil (Figure 1A). During the growth period, water was suctioned from the bottom of the cell tray, water-saving conditions were maintained, and tillers were removed as needed. Egg mass number was counted at 48 days after germination (34 days after inoculation). To count the egg masses, the soil was gently removed from the roots with water and the roots were stained by immersion in 50 ng/µl erioglaucine (Erioglaucine disodium salt, Sigma-Aldrich, #861146) for at least 15 min followed by three water rinses (Figure 1B, C).

And also, the evaluation system using paper pouch is as follows. Rice seeds were soaked in sterilizing solution for reduce the contamination of mold and bacteria, 1000 times dilution of commercial bleach for kitchen (Kao Corporation), for three days at 26°C to induce germination, and then the seeds were sown on a transparent paper pouch (CYG Seed Germination Pouch, Mega International, USA). Two seeds were sown per pouch (Figure 1D). The pouches were customized for stable rice seedlings (Figure 2). The pouches with germinated seeds were placed at 26°C in darkness for 3 days. Then, ten pouches were pressed together between wooden boards with spring clamps (Figure 1E) and placed in light for 12 h at 26°C and in darkness for 12 h at 24°C for 8 days. The paper pouches remained sandwiched between wooden boards throughout the experiment to exclude excess water, as flooded conditions inhibit nematode infection. Fourteen days after the

Figure 1. Evaluation of the soil and paper pouch systems. A–C: soil system. D–K: paper pouch system. A: Inoculation on rice seedlings grown in soil. B: Immersion of the whole root system in 50 ng/µl erioglaucine at 34 days after inoculation (right beaker). After immersion for at least 15 min., the roots were rinsed with water (left beaker). C: Stained egg masses (arrow heads at blue dots). D: Rice seedlings at three days after planting in a paper pouch. E: Paper pouches sandwiched between wooden boards. F, G: Inoculation in the paper pouch system. F: Inoculation was performed along the root (blue areas). G: State of inoculation. The inoculation was performed by attaching a Pasteur pipette to the tip of the p1000 pipettor, widening the opening of a paper pouch, and inserting the Pasteur pipette into it. H–K: Root systems at 34 days after inoculation. H, I: whole root systems. J, K: Magnified images of boxed areas in H and I, respectively. H, J: Before staining. I, K: After staining. Root knots could be confirmed before staining and several egg masses could be confirmed in one knot after staining.

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soaking period, 800 M. incognita individuals at the J2 growth stage were inoculated to each plant along the root by adding 2 ml of a 400 individuals/ml solution (blue box in Figure 1F, G). After inoculation, the sandwiched pouches were laid horizontally in darkness for three days and then placed upright in 12 h light at 28°C and 12 h dark at 26°C. Also, the water-saving conditions were maintained during the growth period, and once a week (7, 14, 21 and 28 days after inoculation), 2 ml of liquid fertilizer (as used in Nishiyama et al. 2015) was added to each plant. Egg mass number was counted at 48 days after germination (34 days after inoculation; Figure 1H–K). The whole root system was stained by immersion in 50 ng/µl erioglaucine for at least 15 min (Figure 1I, K).

For quantification of egg mass production, we counted egg mass numbers in the roots with a stereomicroscope (Leica MZFLIII) at 34 days after inoculation. After counting, the root system was sampled and dried in a 55°C incubator for 5 to 7 days and then the dried root was weighed. And then, we calculated the egg mass number (EMN) per unit of dried root weight (DRW) (EMN/DRW) of each variety. Then, the relative EMN/DRW was calculated against the EMN/DRW of T65, which is susceptible to M. incognita infection. The relative EMN/DRW was used as the evaluation value.

We evaluated 14 rice varieties in two experiment (experiment 1 and experiment 2). The experiment 1 contained cv. T65, Nipponbare, DV85, NERICA 1, NERICA L20, Basmati370, Shoni, and Badari Dhan, and the experiment 2 contained cv. T65, Nipponbare, Hinohikari, Sensho, Kasalath, Kalo Dhan, Shoni, ARC5955, ARC10313, Shoni, and Badari Dhan. The four cultivars, T65, Nipponbare, Shoni and Badari Dhan were contained the both experiments, T65 and Nipponbare as susceptible control, Nipponbare, Shoni and Badari Dhan for confirming reproducibility between experiments. The experiment 1 was evaluated three times using both the soil and the paper pouch system, and the experiment 2 was evaluated once using soil and twice using the paper pouch system. In the soil system, three seeds of each

variety were sown per replicate, and the randomized block method was used to determine the arrangement. In the paper pouch system, four seeds of each variety were sown in two paper pouches per replicate, and the arrangement of pouches in boards was shuffled once a week.

In the experiment 1, T65 and Nipponbare were used as susceptible rice varieties. We also used six rice varieties, DV85, Shoni, Badari Dhan, NERICA 1, NERICA L20, and Basmati370. Nipponbare was more susceptible than T65 to M. incognita, and the other varieties showed different levels of resistance (Figure 3A). Similar infection rates were obtained using the paper pouch and soil systems (Figure 3B). We performed a two-dimensional scatter plot analysis between evaluation values in the soil system and the paper pouch system (Figure 3C). The single regression line was “y=1.246x−0.064”, and the correlation coefficient was 0.965. These values indicated that the nematode infection rate between the soil and paper pouch systems are very similar.

We further compared these evaluation systems using the experiment 2. Hinohikari showed susceptibility similar to T65 and Nipponbare, and seven other varieties showed resistance both in the soil and paper pouch systems (Figure 3D, E). Two-dimensional scatter plot analysis in the experiment 2 showed the single regression straight line “y=1.175x+0.026”, and the correlation coefficient was 0.985 (Figure 3F).

The single regression line obtained when adding the results of the experiments 1 and 2 was “y=1.195x−0.008”, and the correlation coefficient was 0.952. Furthermore, Nipponbare, Shoni, and Badari Dhan dots in both experiments were located close to each other (Figure 3F). These results strongly suggest that the transparent paper pouch system can be used to evaluate nematode susceptibility in various rice varieties with similar outcomes to that of the soil system. In the soil system, Nipponbare was more susceptible than T65 (Figure 3A, C), while in the paper pouch system it was same level as T65 (Figure 3B, D). This tendency also be confirmed between T65 and Hinohikari (Figure 3C, D). In the case of varieties that show high susceptibility in the soil system such as Nipponbare, the paper pouch system may underestimate them. However, this can be resolved by evaluating multiple susceptible varieties as controls.

The development of nematode-induced galls can be observed in the whole root system using our transparent paper pouch system. Specifically, this system can be used to observe the spatial and temporal distribution of developing galls and the growth process of each gall.

Furthermore, this paper pouch system requires only a limited amount of space. We could evaluate 720 rice plants in a single growth chamber (inside diameter;

Figure 2. Customization of the paper pouch for stable rice seedlings. In order not to touch the seedling leaves to the paper, a part of top of the paper was cut off. A and B in front view in customized paper pouches corresponds to A and B in the side view, respectively.

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67 cm wide×110 cm height×56.5 cm depth). In the case of the soil system, at least five growth chambers were needed to grow the same number of rice plants. Thus, the transparent paper pouch system offers a promising new approach to study plant–nematode interactions not only to monitor developing gall in the whole root system over time but also to save the space of growth chamber.

Acknowledgements

We thank National Agriculture and Food Research Organization (NARO) Genebank and Nagoya University, Japan to provide us rice seeds. We also thank to Dr. Kazuyuki Doi for valuable discussions; and Ms. Yasuko Chiba and Ms. Kasumi Dainobu for excellent technical assistance. This work was supported by JSPS KAKENHI (24114009, 24370024, 16K14757, 17H03967 and 18H05487 to S.S.).

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Figure 3. Evaluation of nematode infection rates in the soil and paper systems. A, B: Experiment 1. D, E: Experiment 2. A, D: Evaluation values in the soil system. B, E: Evaluation values in the paper pouch system. C, F: Two-dimensional scatter plots between evaluation values in the soil and paper pouch systems in experiment 1 (C) and in experiments 1 and 2 (F). In panel F, red dots show experiment 1 (Ex 1) and blue dots show experiment 2 (Ex 2), and the two lines show the single regression straight lines in experiment 1 (lower line) and experiment 2 (upper line); the single regression line in experiment 2 was y=1.175x+0.026 and the correlation coefficient was 0.985. Grey (C), red (F), and blue (F) belts show 95% confidence areas; grey and red for experiment 1 and blue for experiment 2. NB, S, and BD indicate Nipponbare, Shoni, and Badari Dhan, respectively, which were evaluated in both experiments for confirming reproducibility. Error bars show standard errors (A–C, E, F).

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