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Cucumber green mottle mosaic virus (CGMMV) can induce hair-like tissues on genus Cucumis seeds
Scientia Horticulturae 146 (2012) 76–80
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Cucumber green mottle mosaic virus (CGMMV) can induce hair-like tissues on genus Cucumis seeds Seung-Yeol Lee a , Nang Kyu Kyu Win a , Dong-Myong Cho b , Su-Heon Lee a , Hee-Young Jung a,∗ a b
School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea Quality Assurance Nong Woo Bio Co., Ltd., Yeoju-Kun, Kyonggi-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 March 2012 Received in revised form 11 August 2012 Accepted 13 August 2012 Keywords: Cucumber green mottle mosaic virus Cucumis seeds Symptom
a b s t r a c t Distinct morphological changes were observed on surface of Cucumber green mottle mosaic virus (CGMMV) infected genus Cucumis seeds. When the infected and healthy seeds were treated with ethanol series, unusual hair-like tissues were observed on surface of infected seeds. Morphology of these tissues was studied by stereo microscopy and field emission scanning electron microscopy. The hair-like tissues were observed on surface of seed coat of infected cucumber, musk melon and oriental melon and raised from the seed coats. Such tissues were not observed on seed coat of the same treated healthy cucumber and musk melon but such tissues were found as attached to seed coat of oriental melons. The width of hairlike tissues varied depend on species of genus Cucumis seeds, 2–2.5 m on musk melon and 9–10 m on oriental melon and cucumber seeds. The phenomenon was re-confirmed by RT-PCR and nested PCR assays that the seeds which had hair-like tissues on their seed coats were infected by CGMMV. Therefore, this study suggests that the hair-like tissues are a unique symptom as external morphological changes of genus Cucumis seeds induced by CGMMV. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Genus Citrullus, Cucumis, Cucurbita and Lagenaria which belong to family Cucurbitaceae include economically important crops and the estimation of their production was over 170 million tons in the world in 2010 (FAOSTAT; http://faostat.fao.org/). Among them, the crops of genus Cucumis such as oriental melon (Cucumis melo L. var. makuwa Makino), musk melon (C. melo L. var. reticulates Naud), cucumber (C. sativus L.), cantaloupe (C. melo L. var. cantalupensis), honeydew (C. melo L. var. inodorus) are widely cultivated all over the world and are actively consumed by cross-border trading. More than 35 viruses infect cucurbits plants, and those belong to genus tobamovirus cause significant economic losses to cucurbit crops (Provvidenti, 1996; Antignus et al., 2001). Cucumber green mottle mosaic virus (CGMMV) is one of the tobamovirus, which is transmitted mechanically and through seeds, and has a narrow host range confined to the Cucurbitaceae (Lee et al., 2011; Varveri et al., 2002). It causes severe mosaic symptoms with discoloration and deformation on plants (Celix et al., 1996; Ali et al., 2004; Kim et al., 2010). Generally, external morphological characters of plant virus infected seeds are discoloration and reduction in seed size (Neergaard, 1997). However, we observed a unique
∗ Corresponding author. Tel.: +82 53 950 5760; fax: +82 53 950 6758. E-mail address: [email protected] (H.-Y. Jung). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.08.016
symptom on CGMMV infected seeds and to our knowledge, such symptom has never been reported before. Therefore, in this study, we presented the unique external symptom induced by CGMMV on infected genus Cucumis seeds with the images of stereo microscopy and FE-SEM.
2. Material and methods 2.1. Seed samples CGMMV infected oriental melon (C. melo L. var. makuwa Makino cv. O-Bok), cucumber (C. sativus L. cv. Dynasty), musk melon (C. melo L. var. reticulates Naud cv. 06MR6001) seeds and each of healthy seeds were collected from experimental field of Nong-Woo Bio Co., Korea, during 2010–2011. Each of seed lots was confirmed for virus infection by reverse transcription-polymerase chain reaction (RTPCR) methods. Thereafter, each of examined seed lots was provided from Nong-Woo Bio Co., Korea. In addition, to verify the effect of experimental condition on seat coat structures, the healthy and infected seeds were dried at 70–75 ◦ C for 7 days or immersed in distilled water for 7 days. These seeds were also examined using stereo microscopy. To examine reproducibility of hair-like tissues observed on seed coat of infected seed, the oriental melon plants were inoculated with CGMMV in restricted area of experimental fields.
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2.2. Detection of CGMMV infected seeds using RT-PCR and nested PCR From provided healthy and CGMMV infected seed lots, each of healthy and infected seeds were randomly sampled and reconfirmed for virus infection by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR methods. Total RNA was extracted by using TRI® reagent solution following to the manufacturer’s protocol. RT-PCR was performed using a AccuPower® RT-PCR Premix (Bioneer, Korea) with CGMMV specific primers; CGMM-C60/CGMM-N30 (5 -AAT TAA GTA AAG TCC TGA CG-3 /5 ATG GAA CGT ACC GGA ATC-3 ) following nested PCR with primers; CGMM-C70/CGMM-N35 (5 -TGG AGG TTT AGA ATG CCG CTT AC3 /5 -ACC GTC CGC GAA TTC TCT-3 ) and the PCR conditions were the same as described previously (Shin, 2009).
2.3. Stereo microscopy and field emission electron microscopy Over 50 seeds from infected and healthy seeds were used to examine. The seeds were immersed in distilled water (DW) for 20 min and then, dehydrated in a graded ethanol series; 30%, 50%, 70%, 80%, 90%, and absolute ethanol for 20 min for each step. Acetone series treatments were also conducted similar to the method as described for ethanol series treatment. After seeds were dried with hexamethyldisilsazane (HMDS) for 15 min, the surface of each treated seed was observed by stereo microscopy (Dimis-M, Siwon optical, Korea). For FE-SEM observation, seeds were fixed and dehydrated following to previously described procedure (Dita et al., 2007). Thereafter, the treated seeds were coated with platinum (30 nm in thickness) using platinum ion coater (E-1030, HITACHI, Japan), and examined with the field emission scanning electron microscopy (FE-SEM, S-4300, HITACHI, Japan) under low (×500) and high (×1500) magnification. Heat dried and water immersed seeds were equally treated with DW, ethanol series and HMDS as described above. Thereafter, the surfaces of seeds were observed by stereo microscopy (Dimis-M, Siwon optical, Korea). The CGMMV infection on examined seeds were re-examined by RT-PCR assay as described above.
Fig. 1. Detection of CGMMV infected seed by nested PCR methods. Amplified DNA fragments were observed by electrophoresis on 0.7% agarose gel. M: 100 bp DNA ladder. Cu, cucumber; Me, musk melon; Or, oriental melon.
3. Results and discussion Each of healthy and CGMMV infected seeds were re-examined CGMMV infection from each seed lots by RT-PCR following nested PCR methods. About 370 bp DNA product was amplified from the infected seeds and no product was amplified from healthy ones (Fig. 1). Based on the PCR results, it was assumed that each of CGMMV infected seeds from provided seed lot were infected by CGMMV and healthy seeds were not infected by CGMMV. Thus, we randomly sampled seed from each seed lots to study the external morphological character. After treating with ethanol series on infected and healthy seeds, hair-like tissues were observed on the surface of seed coats of infected cucumber, musk melon and oriental melon (Fig. 2D–F) and such tissues were not observed on those of healthy ones (Fig. 2A–C). Furthermore, hair-like tissues were observed all the examined infected seeds while any changes were observed on healthy seeds. The hair-like tissues were found on the entire seed coat of cucumber and oriental melon (Fig. 2D and F), and locally on those of musk melon seeds (Fig. 2E). Besides, the presence of hair-like tissues on CGMMV infected seeds was also observed despite both the infected and healthy seeds were treated with acetone series (Fig. 3). Based on the results of nested PCR re-examination, it was confirmed that the hair-like tissues on infected seeds were induced by CGMMV infection.
Fig. 2. Stereo micrographs of healthy (upper) and CGMMV infected (lower) seeds after treated with ethanol series. (A and D) Cucumber. (B and E) Musk melon. (C and F) Oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 500 m.
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Fig. 3. Stereo micrographs of healthy (upper) and CGMMV infected (lower) seeds after treated with acetone series. (A and D) Cucumber. (B and E) Musk melon. (C and F) Oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 500 m.
The detail structures of hair-like tissues were studied by using FE-SEM under low and high magnification (Fig. 4). The hair-like tissues were found as raised structures on infected seed coats. Although the hair-like tissues were also found on healthy oriental melon seeds (Fig. 4I), these tissues were not detached from the seed coat (Fig. 4J). Depend on genus Cucumis species, the width of hair-like tissue varied. The hair-like tissues on infected musk melon seeds were 2–2.5 m in width and those on infected cucumber and oriental melon were 9–10 m. These morphological variations might be due to the examined seeds which belong to different species of genus Cucumis.
The induction of hair-like tissues by CGMMV was further investigated on heat dried or water immersed infected seeds and the artificially CGMMV inoculated oriental melon seeds. The seeds treated with two experimental conditions also showed the hairlike tissues on seed coats (Figs. 5 and 6). The results proofed that the appearance of hair-like tissues on infected seeds was not due to other environmental conditions such as drying or immersing in water. Also, the oriental melon seeds collected from the inoculated plants were treated with ethanol and acetone series as described above. The same hair-like tissues were observed on seed coats of both ethanol and acetone treated seeds (Fig. 7A and B).
Fig. 4. Field emission scanning electron micrographs of healthy and CGMMV infected seeds. (A and B) Healthy cucumber. (C and D) Infected cucumber. (E, F) Healthy musk melon. (G and H) Infected musk melon. (I and J) Healthy oriental melon. (K and L) Infected oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 20 m. (Low magnification (×500): A, C, E, G, I and K; high magnification (×1500): B, D, F, H, J and L.)
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Fig. 5. Stereo micrographs of heat dried healthy (upper) and CGMMV infected (lower) seeds for 7 days after treated with ethanol series. (A and D) Cucumber. (B and E) Musk melon. (C and F) Oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 500 m.
Generally, external symptoms of virus infected plants are mosaic, mottle, yellowing and deformation (Zechmann et al., 2003). Consequently, virus infected seeds, deformation and discoloration and all these symptoms can be seen by naked eyes. For example, wrinkled seed coat and discolored symptoms were on Pea early browning virus infected pea (Pisum sativum L.). Likewise, Mung bean mosaic virus infected mung bean (Vigna radiata L.) can be characterized by reduced seed size and discolored seed size, when compared to healthy seeds (Neergaard, 1997). Desbiez and Lecoq (1997) reported that Cucumis seeds became deformed and smaller than healthy seeds when musk melon seeds were infected by Zucchini yellow mosaic virus (Desbiez and Lecoq, 1997). Recently, we have reported the internal changes on CGMMV infected oriental
melon seeds compared with healthy seeds (Lee et al., 2011). However, until now, there was no report about external changes of seed-borne virus infected seeds except deformation, discoloration and size reduction. In this study, the examined seeds could not be separated by naked eyes into infected or non-infected seeds. However, we could differentiate the infected seeds by checking the presence of hair-like tissues and its structure with the aid of stereo microscopy after ethanol treatment. Moreover, all the examined CGMMV infected genus Cucumis seeds showed hair-like tissues on the seed coat. Although reproducibility tests are still needed, we expected that this easy method can be used as differentiation method between healthy and CGMMV infected seeds. Furthermore, plant physiological studies are still needed to understand
Fig. 6. Stereo micrographs of water immersed healthy (upper) and CGMMV infected (lower) seeds for 7 days after treated with ethanol series. (A and D) Cucumber. (B and E) Musk melon. (C and F) Oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 500 m.
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Fig. 7. Stereo micrographs of CGMMV inoculated oriental melon seeds after treated with ethanol (A) and acetone (B) series. Scale bar: 500 m.
the appearance of unique symptom, which is induced by CGMMV infection. Acknowledgements This research was supported by Export Promotion Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. References Ali, A., Natsuaki, T., Okuda, S., 2004. Identification and molecular characterization of viruses infecting cucurbits in Pakistan. J. Phytopathol. 152, 677–682. Antignus, Y., Wang, Y., Pearlsman, M., Lachman, O., Lavi, N., Gal-On, A., 2001. Biological and molecular characterization of a unique cucurbit infection tobamovirus. Phytopathology 91, 565–571. Celix, A., Luis-Arteaga, M., Rodriguez-Cerezo, E., 1996. First report of Cucumber green mottle mosaic tobamovirus infecting greenhouse-grown cucumber in Spain. Plant Dis. 80, 1303. Desbiez, C., Lecoq, H., 1997. Zucchini yellow mosaic virus. Plant Pathol. 46, 809–829.
Dita, M.A., Brommonschenkel, S.H., Matsuoka, K., Mizubuti, E.S.G., 2007. Histopathological study of the Alternaria solani infection process in potato cultivars with different levels of early blight resistance. J. Phytopathol. 155, 462–469. Kim, O.K., Mizutani, T., Natsuaki, T., Lee, K.W., Soe, K., 2010. First report and the genetic variability of Cucumber green mottle mosaic virus occurring on bottle gourd in Myanmar. J. Phytopathol. 158, 572–575. Lee, C.H., Lee, S.Y., Kim, J.Y., Jung, H.Y., Kim, J.H., 2011. Optical sensing method for screening disease in melon seeds using optical coherence tomography. Sensors 11, 9467–9477. Neergaard, P., 1997. Diseases and injuries of seeds. Seed Pathology, vol. I. The Gresham Press, Old woking, Surrey, England, pp. 49–51. Provvidenti, R., 1996. Diseases caused by viruses. In: Zitter, T.A., Hopkins, D.L., Thomas, C.E. (Eds.), Compendium of Cucurbit Diseases. APS Press, St. Paul, MN, USA, pp. 37–45. Shin, Y.G., 2009. An advanced quarantine system for the inspection of seed-borne viruses in Korea. Ph.D. Thesis. Kyungpook National University, Daegu, Korea (in Korean). Varveri, C., Vassilakos, N., Bem, F., 2002. Characterization and detection of cucumber green mottle mosaic virus in Greece. Phytoparasitica 30, 493–501. Zechmann, B., Müller, M., Zellnig, G., 2003. Cytological modification in zucchini yellow mosaic virus (ZYMV)-infected Stryrian pumpkin plants. Arch. Virol. 148, 1119–1133.
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Cucumber green mottle mosaic virus (CGMMV) can induce hair-like tissues on genus Cucumis seeds Seung-Yeol Lee a , Nang Kyu Kyu Win a , Dong-Myong Cho b , Su-Heon Lee a , Hee-Young Jung a,∗ a b
School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea Quality Assurance Nong Woo Bio Co., Ltd., Yeoju-Kun, Kyonggi-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 March 2012 Received in revised form 11 August 2012 Accepted 13 August 2012 Keywords: Cucumber green mottle mosaic virus Cucumis seeds Symptom
a b s t r a c t Distinct morphological changes were observed on surface of Cucumber green mottle mosaic virus (CGMMV) infected genus Cucumis seeds. When the infected and healthy seeds were treated with ethanol series, unusual hair-like tissues were observed on surface of infected seeds. Morphology of these tissues was studied by stereo microscopy and field emission scanning electron microscopy. The hair-like tissues were observed on surface of seed coat of infected cucumber, musk melon and oriental melon and raised from the seed coats. Such tissues were not observed on seed coat of the same treated healthy cucumber and musk melon but such tissues were found as attached to seed coat of oriental melons. The width of hairlike tissues varied depend on species of genus Cucumis seeds, 2–2.5 m on musk melon and 9–10 m on oriental melon and cucumber seeds. The phenomenon was re-confirmed by RT-PCR and nested PCR assays that the seeds which had hair-like tissues on their seed coats were infected by CGMMV. Therefore, this study suggests that the hair-like tissues are a unique symptom as external morphological changes of genus Cucumis seeds induced by CGMMV. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Genus Citrullus, Cucumis, Cucurbita and Lagenaria which belong to family Cucurbitaceae include economically important crops and the estimation of their production was over 170 million tons in the world in 2010 (FAOSTAT; http://faostat.fao.org/). Among them, the crops of genus Cucumis such as oriental melon (Cucumis melo L. var. makuwa Makino), musk melon (C. melo L. var. reticulates Naud), cucumber (C. sativus L.), cantaloupe (C. melo L. var. cantalupensis), honeydew (C. melo L. var. inodorus) are widely cultivated all over the world and are actively consumed by cross-border trading. More than 35 viruses infect cucurbits plants, and those belong to genus tobamovirus cause significant economic losses to cucurbit crops (Provvidenti, 1996; Antignus et al., 2001). Cucumber green mottle mosaic virus (CGMMV) is one of the tobamovirus, which is transmitted mechanically and through seeds, and has a narrow host range confined to the Cucurbitaceae (Lee et al., 2011; Varveri et al., 2002). It causes severe mosaic symptoms with discoloration and deformation on plants (Celix et al., 1996; Ali et al., 2004; Kim et al., 2010). Generally, external morphological characters of plant virus infected seeds are discoloration and reduction in seed size (Neergaard, 1997). However, we observed a unique
∗ Corresponding author. Tel.: +82 53 950 5760; fax: +82 53 950 6758. E-mail address: [email protected] (H.-Y. Jung). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.08.016
symptom on CGMMV infected seeds and to our knowledge, such symptom has never been reported before. Therefore, in this study, we presented the unique external symptom induced by CGMMV on infected genus Cucumis seeds with the images of stereo microscopy and FE-SEM.
2. Material and methods 2.1. Seed samples CGMMV infected oriental melon (C. melo L. var. makuwa Makino cv. O-Bok), cucumber (C. sativus L. cv. Dynasty), musk melon (C. melo L. var. reticulates Naud cv. 06MR6001) seeds and each of healthy seeds were collected from experimental field of Nong-Woo Bio Co., Korea, during 2010–2011. Each of seed lots was confirmed for virus infection by reverse transcription-polymerase chain reaction (RTPCR) methods. Thereafter, each of examined seed lots was provided from Nong-Woo Bio Co., Korea. In addition, to verify the effect of experimental condition on seat coat structures, the healthy and infected seeds were dried at 70–75 ◦ C for 7 days or immersed in distilled water for 7 days. These seeds were also examined using stereo microscopy. To examine reproducibility of hair-like tissues observed on seed coat of infected seed, the oriental melon plants were inoculated with CGMMV in restricted area of experimental fields.
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2.2. Detection of CGMMV infected seeds using RT-PCR and nested PCR From provided healthy and CGMMV infected seed lots, each of healthy and infected seeds were randomly sampled and reconfirmed for virus infection by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR methods. Total RNA was extracted by using TRI® reagent solution following to the manufacturer’s protocol. RT-PCR was performed using a AccuPower® RT-PCR Premix (Bioneer, Korea) with CGMMV specific primers; CGMM-C60/CGMM-N30 (5 -AAT TAA GTA AAG TCC TGA CG-3 /5 ATG GAA CGT ACC GGA ATC-3 ) following nested PCR with primers; CGMM-C70/CGMM-N35 (5 -TGG AGG TTT AGA ATG CCG CTT AC3 /5 -ACC GTC CGC GAA TTC TCT-3 ) and the PCR conditions were the same as described previously (Shin, 2009).
2.3. Stereo microscopy and field emission electron microscopy Over 50 seeds from infected and healthy seeds were used to examine. The seeds were immersed in distilled water (DW) for 20 min and then, dehydrated in a graded ethanol series; 30%, 50%, 70%, 80%, 90%, and absolute ethanol for 20 min for each step. Acetone series treatments were also conducted similar to the method as described for ethanol series treatment. After seeds were dried with hexamethyldisilsazane (HMDS) for 15 min, the surface of each treated seed was observed by stereo microscopy (Dimis-M, Siwon optical, Korea). For FE-SEM observation, seeds were fixed and dehydrated following to previously described procedure (Dita et al., 2007). Thereafter, the treated seeds were coated with platinum (30 nm in thickness) using platinum ion coater (E-1030, HITACHI, Japan), and examined with the field emission scanning electron microscopy (FE-SEM, S-4300, HITACHI, Japan) under low (×500) and high (×1500) magnification. Heat dried and water immersed seeds were equally treated with DW, ethanol series and HMDS as described above. Thereafter, the surfaces of seeds were observed by stereo microscopy (Dimis-M, Siwon optical, Korea). The CGMMV infection on examined seeds were re-examined by RT-PCR assay as described above.
Fig. 1. Detection of CGMMV infected seed by nested PCR methods. Amplified DNA fragments were observed by electrophoresis on 0.7% agarose gel. M: 100 bp DNA ladder. Cu, cucumber; Me, musk melon; Or, oriental melon.
3. Results and discussion Each of healthy and CGMMV infected seeds were re-examined CGMMV infection from each seed lots by RT-PCR following nested PCR methods. About 370 bp DNA product was amplified from the infected seeds and no product was amplified from healthy ones (Fig. 1). Based on the PCR results, it was assumed that each of CGMMV infected seeds from provided seed lot were infected by CGMMV and healthy seeds were not infected by CGMMV. Thus, we randomly sampled seed from each seed lots to study the external morphological character. After treating with ethanol series on infected and healthy seeds, hair-like tissues were observed on the surface of seed coats of infected cucumber, musk melon and oriental melon (Fig. 2D–F) and such tissues were not observed on those of healthy ones (Fig. 2A–C). Furthermore, hair-like tissues were observed all the examined infected seeds while any changes were observed on healthy seeds. The hair-like tissues were found on the entire seed coat of cucumber and oriental melon (Fig. 2D and F), and locally on those of musk melon seeds (Fig. 2E). Besides, the presence of hair-like tissues on CGMMV infected seeds was also observed despite both the infected and healthy seeds were treated with acetone series (Fig. 3). Based on the results of nested PCR re-examination, it was confirmed that the hair-like tissues on infected seeds were induced by CGMMV infection.
Fig. 2. Stereo micrographs of healthy (upper) and CGMMV infected (lower) seeds after treated with ethanol series. (A and D) Cucumber. (B and E) Musk melon. (C and F) Oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 500 m.
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Fig. 3. Stereo micrographs of healthy (upper) and CGMMV infected (lower) seeds after treated with acetone series. (A and D) Cucumber. (B and E) Musk melon. (C and F) Oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 500 m.
The detail structures of hair-like tissues were studied by using FE-SEM under low and high magnification (Fig. 4). The hair-like tissues were found as raised structures on infected seed coats. Although the hair-like tissues were also found on healthy oriental melon seeds (Fig. 4I), these tissues were not detached from the seed coat (Fig. 4J). Depend on genus Cucumis species, the width of hair-like tissue varied. The hair-like tissues on infected musk melon seeds were 2–2.5 m in width and those on infected cucumber and oriental melon were 9–10 m. These morphological variations might be due to the examined seeds which belong to different species of genus Cucumis.
The induction of hair-like tissues by CGMMV was further investigated on heat dried or water immersed infected seeds and the artificially CGMMV inoculated oriental melon seeds. The seeds treated with two experimental conditions also showed the hairlike tissues on seed coats (Figs. 5 and 6). The results proofed that the appearance of hair-like tissues on infected seeds was not due to other environmental conditions such as drying or immersing in water. Also, the oriental melon seeds collected from the inoculated plants were treated with ethanol and acetone series as described above. The same hair-like tissues were observed on seed coats of both ethanol and acetone treated seeds (Fig. 7A and B).
Fig. 4. Field emission scanning electron micrographs of healthy and CGMMV infected seeds. (A and B) Healthy cucumber. (C and D) Infected cucumber. (E, F) Healthy musk melon. (G and H) Infected musk melon. (I and J) Healthy oriental melon. (K and L) Infected oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 20 m. (Low magnification (×500): A, C, E, G, I and K; high magnification (×1500): B, D, F, H, J and L.)
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Fig. 5. Stereo micrographs of heat dried healthy (upper) and CGMMV infected (lower) seeds for 7 days after treated with ethanol series. (A and D) Cucumber. (B and E) Musk melon. (C and F) Oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 500 m.
Generally, external symptoms of virus infected plants are mosaic, mottle, yellowing and deformation (Zechmann et al., 2003). Consequently, virus infected seeds, deformation and discoloration and all these symptoms can be seen by naked eyes. For example, wrinkled seed coat and discolored symptoms were on Pea early browning virus infected pea (Pisum sativum L.). Likewise, Mung bean mosaic virus infected mung bean (Vigna radiata L.) can be characterized by reduced seed size and discolored seed size, when compared to healthy seeds (Neergaard, 1997). Desbiez and Lecoq (1997) reported that Cucumis seeds became deformed and smaller than healthy seeds when musk melon seeds were infected by Zucchini yellow mosaic virus (Desbiez and Lecoq, 1997). Recently, we have reported the internal changes on CGMMV infected oriental
melon seeds compared with healthy seeds (Lee et al., 2011). However, until now, there was no report about external changes of seed-borne virus infected seeds except deformation, discoloration and size reduction. In this study, the examined seeds could not be separated by naked eyes into infected or non-infected seeds. However, we could differentiate the infected seeds by checking the presence of hair-like tissues and its structure with the aid of stereo microscopy after ethanol treatment. Moreover, all the examined CGMMV infected genus Cucumis seeds showed hair-like tissues on the seed coat. Although reproducibility tests are still needed, we expected that this easy method can be used as differentiation method between healthy and CGMMV infected seeds. Furthermore, plant physiological studies are still needed to understand
Fig. 6. Stereo micrographs of water immersed healthy (upper) and CGMMV infected (lower) seeds for 7 days after treated with ethanol series. (A and D) Cucumber. (B and E) Musk melon. (C and F) Oriental melon. Arrow head: indication of hair-like tissues. Scale bar: 500 m.
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Fig. 7. Stereo micrographs of CGMMV inoculated oriental melon seeds after treated with ethanol (A) and acetone (B) series. Scale bar: 500 m.
the appearance of unique symptom, which is induced by CGMMV infection. Acknowledgements This research was supported by Export Promotion Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. References Ali, A., Natsuaki, T., Okuda, S., 2004. Identification and molecular characterization of viruses infecting cucurbits in Pakistan. J. Phytopathol. 152, 677–682. Antignus, Y., Wang, Y., Pearlsman, M., Lachman, O., Lavi, N., Gal-On, A., 2001. Biological and molecular characterization of a unique cucurbit infection tobamovirus. Phytopathology 91, 565–571. Celix, A., Luis-Arteaga, M., Rodriguez-Cerezo, E., 1996. First report of Cucumber green mottle mosaic tobamovirus infecting greenhouse-grown cucumber in Spain. Plant Dis. 80, 1303. Desbiez, C., Lecoq, H., 1997. Zucchini yellow mosaic virus. Plant Pathol. 46, 809–829.
Dita, M.A., Brommonschenkel, S.H., Matsuoka, K., Mizubuti, E.S.G., 2007. Histopathological study of the Alternaria solani infection process in potato cultivars with different levels of early blight resistance. J. Phytopathol. 155, 462–469. Kim, O.K., Mizutani, T., Natsuaki, T., Lee, K.W., Soe, K., 2010. First report and the genetic variability of Cucumber green mottle mosaic virus occurring on bottle gourd in Myanmar. J. Phytopathol. 158, 572–575. Lee, C.H., Lee, S.Y., Kim, J.Y., Jung, H.Y., Kim, J.H., 2011. Optical sensing method for screening disease in melon seeds using optical coherence tomography. Sensors 11, 9467–9477. Neergaard, P., 1997. Diseases and injuries of seeds. Seed Pathology, vol. I. The Gresham Press, Old woking, Surrey, England, pp. 49–51. Provvidenti, R., 1996. Diseases caused by viruses. In: Zitter, T.A., Hopkins, D.L., Thomas, C.E. (Eds.), Compendium of Cucurbit Diseases. APS Press, St. Paul, MN, USA, pp. 37–45. Shin, Y.G., 2009. An advanced quarantine system for the inspection of seed-borne viruses in Korea. Ph.D. Thesis. Kyungpook National University, Daegu, Korea (in Korean). Varveri, C., Vassilakos, N., Bem, F., 2002. Characterization and detection of cucumber green mottle mosaic virus in Greece. Phytoparasitica 30, 493–501. Zechmann, B., Müller, M., Zellnig, G., 2003. Cytological modification in zucchini yellow mosaic virus (ZYMV)-infected Stryrian pumpkin plants. Arch. Virol. 148, 1119–1133.