Survey of Nematodes in Coniferous Bonsai in Korea

Article information

Res. Plant Dis. 2016;22(4):243-248
Publication date (electronic) : 2016 December 31
doi : https://doi.org/10.5423/RPD.2016.22.4.243
1 Department of Plant Bioscience, Pusan National University, Miryang 50463, Korea
2 Nematode Research Center, Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Korea
3 Plant Quarantine Technology Research & Development Center, Department of Plant Quarantine, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
*Corresponding author Tel: +82-55-350-5692 Fax: +82-55-350-5509 E-mail: ichoi@pusan.ac.kr

These authors contributed equally to this work as co-first authors.

Received 2016 August 11; Revised 2016 October 14; Accepted 2016 October 24.

Abstract

As preventive measures for bonsai exports, nematodes were isolated from 55 bonsai samples of five coniferous species (Chamaecyparis pisifera, Juniperus chinensis, Pinus densiflora, Pinus thunbergii, and Taxus cuspidate) from all 15 bonsai gardens in Korea. Nematodes belonging to 21 genera in 7 orders were isolated from the 55 bonsai samples. Among plant-parasitic nematodes, Tylenchus spp. was the most frequently isolated (14.9%), followed by Ditylenchus spp. (10.5%), Aphelenchoides spp. (9.5%), Aphelenchus sp. (5.5%), Criconemoides sp. (4.0%), Helicotylenchus sp. (0.7%), Hemicycliophora sp. (0.7%), Mesocriconema sp. (0.7%), Tylenchorhynchus sp. (0.7%), and Paratylenchus sp. (0.4%). Among non-parasitic nematodes, Cephalobina was the most frequently isolated nematodes (26.5%), followed by Rhabditida (19.3%), Dorylaimida (17.8%), Pangrolaimida (14.5%), Plectida (6.5%), Tryphylida (6.2%), Mononchida (3.3%), Alaimida (2.9%), Monhysterida (2.5%), and Triplonchida (0.4%). Based on these results, we conclude that there is no problematic plant-parasitic nematode in bonsai gardens of Korea.

Introduction

Bonsai refers to trees growing in pots for people who enjoy watching them. It is considered as an elegant hobby for generations. Today, bonsai is an export commodity. It has become a high-value product. Japanese bonsai is renowned worldwide. However, Japanese bonsai is suffering due to aged technicians, high-wages, and avoidance of labor-intensive growing for bonsai by young people. China is supplying a huge amount of low quality bonsai to the world market based on large acreage and low wages. If Korean bonsai gardeners could supply high quality bonsai at affordable prices, they could have a competitive edge in the export market of bonsai.

For bonsai exports, EU and the United States require that bonsai should be grown in certified gardens that meet their specific standards. Recently, EU and other countries have strengthened nematode inspection for bonsai during the quarantine process. To promote bonsai exports, bonsai should be free of plant-parasitic nematodes. There are 1,500 plant-parasitic nematodes. They cause crop losses with estimated economic value of about $1,180 billion worldwide. In Korea, 132 species in 42 genera and 12 families of plant-parasitic nematodes have been reported (Choi, 2001). However, bonsai nematodes have not been reported yet. Therefore, the objective of this study was to survey nematodes in bonsai gardens of Korea.

Materials and Methods

Bonsai collections

The study is focused on coniferous bonsai because they are the main export commodities. We collected 55 bonsai samples in five coniferous species from 15 bonsai gardens (Table 1). The five species were: Juniperus chinensis, Pinus densiflora, Pinus thunbergii, Taxus cuspidata, and Chamaecyparis pisifera. To investigate the source of nematode contamination, bonsai culture medium and water sources were also examined (Fig. 1, Table 1). Samples were collected from July to September when nematode populations were abundant.

Sampling area, Bonsai species, and number of bonsai samples used in this study

Fig. 1

Location of bonsai gardens for nematode samplings (Goyang, Seosan, Chungwon, Daejeon, Okcheon, Muan, Yeosu, Changwon, Busan, Daegu, Gyeongsan, Pohang).

Nematode isolation and slide preparation

Root, leaf, stem, and soil samples were prepared. Roots, leaves, and stems were cut into size of 1 cm2 and placed in tap water for 24 hours. After filtering through 200 and 500 mesh sieves, debris were collected from 500 mesh sieves and further processed using Baermann’s funnel method (Southey, 1986). Criconematidae are slow moving nematodes. They could not be isolated by Baermann’s funnel. Therefore, centrifugal flotation method was used to isolate Criconematid nematodes (Whitehead and Hemming, 1965).

To detect nematodes from water sources used in bonsai gardens, 20 l water was collected from each bonsai garden and filter with 500 mesh sieves. Nematodes were collected from 500 mesh sieves and examined under a microscope.

To detect nematodes from growing medium used in bonsai gardens, 300 g of growing medium collected from each bonsai garden was placed in water for 24 hours. Nematodes were then isolated by using 200 and 500 mesh sieves.

Isolated nematodes were fixed with hot formaldehyde-glycerol 4:1 solution and then transferred to glycerin according to Seinhorst’s rapid method (Seinhorst, 1959). Slides were examined under an Olympus BX53 microscope (Olympus, Tokyo, Japan). Plant-parasitic nematodes are important in quarantine. Therefore, they were identified to the species level. Non-plant-parasitic nematodes were identified to the order level. Recently nematode classification system, especially in higher ranks, has been changed significantly due to the progress of molecular research (Nemaplex, 1999). Nematode taxonomy has been strongly based on morphological characters (De Ley et al., 2005) as the traditional classification system (Winvertebrates, 2015). Such traditional classification system is more appropriate for nematodes found in bonsai compared to molecular approaches. Therefore, we used the traditional classification system for this study.

Results and Discussion

Isolated nematodes

Nematodes were isolated from all 15 bonsai gardens and 55 coniferous bonsai species. They belonged to 21 genera in 7 orders (Table 2).

Nematodes isolated from coniferous bonsai in 2015*

Among Tylechida, Tylenchus was the most frequently identified (14.9%), followed by Ditylenchus (10.5%), Criconemoides (4.0%), Helicotylenchus (0.7%), Hemicycliophora (0.7%), Mesocriconema (0.7%), Tylenchorhynchus (0.7%), and Paratylenchus (0.4%). Most Tylenchus and Ditylenchus are known as fungivorous.

Among Aphelenchida, Aphelenchoides (9.5%) and Aphelenchus (5.5%) were isolated. Except for few species of Aphelenchoides, most Aphelenchoides were fungivorous or insect-parasitic nematodes. Aphelenchus is a well-known fungivorous nematode. They are not problematic nematodes for exports.

Non-parasitic nematodes included Cephalobina (26.5%), Rhabditida (19.3%), Dorylaimida (17.8%), Pangrolaimida (14.5%), Plectida (6.5%), Tryphylida (6.2%), Mononchida (3.3%), Alaimida (2.9%), Monhysterida (2.5%), and Triplonchida (0.4%) (Table 2). These non-plant-parasitic nematodes are abundant in all natural environments in the world.

Nematode from different parts of bonsai

When roots, stems, leaves, and soil samples (Baermann’s funnel method and Centrifugal flotation method) were examined, nematodes were more frequently found from roots (10.3%). They were less frequently found in stems (2.2%) or leaves (2.2%).

Among plant-parasitic nematodes isolated from roots, Tylenchus (30.9%) and Ditylenchus (29.1%) were the more frequently found ones, followed by Aphelenchoides parasaprophilus (16.4%), Aphelenchus avenae (14.5%), Criconemoides annulatus (1.8%), and Helicotylenchus pseudorobustus (1.8%). A. parasaprophilus, A. avenae, and Ditylenchus were also isolated from leaves and stems.

Non-parasitic nematodes isolated from roots were Cephalobina (41.8%), Dorylaimida (30.9%), Rhabditida (20.0%), Pangrolaimida (18.2%), Alaimida (1.8%), Monhysterida (3.6%), and Mononchida (3.6%) (Table 2).

For soil isolation, Baermann’s funnel method (11.7%) isolated more nematodes than the centrifugal flotation method (9.2%). The following four new nematode genera were isolated from soil only: Hemicycliophora (3.6%), Mesocriconema (3.6%), Paratylenchus (1.8%), and Tylenchorhynchus crassicadatus (1.8%). In soil isolation, Tylenchus and Ditylenchus were the most frequently isolated nematodes (25.5% and 10.9%, respectively). Mesocriconema and Paratylenchus were only isolated by the centrifugal flotation method. C. annulatus was recovered four times more in numbers compared to that by the Baermann’s funnel method (Table 2). Non-parasitic nematodes isolated from soil samples were Cephalobina (40.0%–43.6%), Dorylaimida (14.5%–38.2%), Rhabditida (27.3%–38.2%), Tryphylida (5.5%–23.6%), Plectida (9.1%–18.1%), Mononchida (12.7%), and Pangrolaimida (10.9%–14.5%) (Table 2).

Nematodes from different bonsai garden

There was no significant difference in nematode numbers among the six provinces (data not shown). Among plant-parasitic nematodes, Criconemoides (5.0%) and Helicotylenchus (5.0%) were isolated from Chungbuk province while Criconemoides (9.1%), Hemicycliophora (3.6%), Mesocriconema (1.8%), Paratylenchus (1.8%), Tylenchorhynchus (3.6%), and Tylenchus (34.5%) were isolated from Gyeongbuk province. Important plant-parasitic nematode was isolated from only one bonsai garden. Its soil was used as growing medium.

Nematodes from different bonsai species

The five bonsai species had different nematode species (data not shown). Since the number of samples ranged from 1 to 27, it was inappropriate to distinguish nematodes by coniferous species.

Nematodes in water source and growing medium used for bonsai gardens

When the growing media collected from bonsai gardens were examined, nematodes were detected from four bonsai gardens (SB garden, CD garden, HR garden, and BJ garden) (Table 3). Various species of non-parasitic nematodes were found from growing media. However, there was no plant-parasitic nematode (Table 3). During the survey, we found that growing medium was left opened in the yard. This could cause nematode contamination. Therefore, growing media should be kept in separate storage facilities after being opened.

Nematode detected from bonsai culture media

When water sources used for bonsai garden were examined, only Aphelenchoides (5 nematodes per 2 liters of water) was detected from SR bonsai garden which used nearby riverlet as water source. Other bonsai gardens that used tap water or underground water as water source did not have nematodes.

During the survey of nematodes in bonsai gardens of Korea, nematodes were mostly isolated from roots and soil samples. Most nematodes were non-parasitic nematodes. Only one bonsai garden was infested with plant-parasitic nematodes. It used soil as growing medium. Therefore, bonsai gardens for exports must use certified growing medium. They should store growing media in separate storage facilities after the bag is opened. When bonsai gardens used underground water or tap water as water supply and bonsai roots are washed to be free of soil before exports, there should be no nematode problem for bonsai to be exported.

References

Choi Y.E. 2001. Economic Insects of Korea 20 Tylenchida, Aphelenchida (Nematoda). Insecta Koreana Suppl 27National Institute of Agricultural Science and Technology. Suwon, Korea: p. 391.
De Ley P, De Ley I.T, Morris K, Abebe E, Mundo-Ocampo M, Yoder M, Heras J, Waumann D, Rocha-Olivares A, Jay Burr AH, Baldwin J.G, Thomas W.K. 2005;An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. Philos. Trans. R. Soc. Lond. B Biol. Sci 360:1945–1958. 10.1098/rstb.2005.1726. 16214752. PMC1609217.
Nemaplex. 1999 onwards. The “Nematode-Plant Expert Information System”. A Virtual Encyclopedia on Soil and Plant Nematodes 20 April 2015. URL http://plpnemweb.ucdavis.edu/nemaplex .
Seinhorst J.W. 1959;A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4:67–69. 10.1163/187529259X00381.
Southey J.F. 1986. Laboratory Methods for Work with Plant and Soil Nematodes. Reference Book (Great Britain. Ministry of Agriculture, Fisheries and Food) 402Her Majesty’s Stationery Office; London, UK: 202. 3800792.
Whitehead A.G, Hemming J.R. 1965;A comparison of some quantitative methods of extracting small vermiform nematodes from soil. Ann. Appl. Biol. J 55:25–38. 10.1111/j.1744-7348.1965.tb07864.x.
2015 onwards. Winvertebrates. 5 June 2015. URL http://winvertebrates.uwsp.edu/Nematoda.html .

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Article information Continued

Table 1

Sampling area, Bonsai species, and number of bonsai samples used in this study

  Province City Bonsai garden* Bonsai species No. of bonsai
sampled
Gyeonggi Goyang SY Juniper (Juniperus chinensis) 3
Gyeongnam Busan SR Juniper (J. chinensis) 3
Black pine (Pinus thunbergii) 2
SB Juniper (J. chinensis) 3
Black pine (P. thunbergii) 1
Changwon HR Juniper (J. chinensis) 2
Black pine (P. thunbergii) 2
GA Juniper (J. chinensis) 1
Black pine (P. thunbergii) 1
Gyeongbuk Gyeongsan CD Juniper (J. chinensis) 3
Black pine (P. thunbergii) 3
Pohang GR Juniper (J. chinensis) 1
Daegu SW Black pine (P. thunbergii) 2
Juniper (J. chinensis) 2
Jeonnam Yeosu BJ Juniper (J. chinensis) 1
Black pine (P. thunbergii) 1
Yew (Taxus cuspidata) 1
Muan GS Black pine (P. thunbergii) 4
Chungnam Seosan UJ Sawara cypress (Chamaecyparis pisifera) 1
CE Black pine (P. thunbergii) 2
Juniper (J. chinensis) 2
Black pine (P. thunbergii) 2
Daejeon HG Juniper (J. chinensis) 2
Pine (Pinus densiflora) 2
Juniper (J. chinensis) 2
Chungbuk Chungwon OS Black pine (P. thunbergii) 3
Okcheon SS Juniper (J. chinensis) 2
Black pine (P. thunbergii) 1
Total 12 15 28 55
*

Initials of bonsai garden.

Fig. 1

Location of bonsai gardens for nematode samplings (Goyang, Seosan, Chungwon, Daejeon, Okcheon, Muan, Yeosu, Changwon, Busan, Daegu, Gyeongsan, Pohang).

Table 2

Nematodes isolated from coniferous bonsai in 2015*

   Nematodes Frequency (%) Average

Plant Soil


Root Leaf Stem Sieve Centrifugal
Plant-parasitic nematodes
 Aphelenchida
  Aphelenchus avenae 14.5 1.8 1.8 3.6 5.5 5.5
  Aphelenchoides parasaprophilus 16.4 12.7 5.5 7.3 5.5 9.5
 Tylenchida
  Criconemoides annulatus 1.8 - - 3.6 14.5 4.0
  Ditylenchus spp. 29.1 3.6 3.6 10.9 5.5 10.5
  Helicotylenchus pseudorobustus 1.8 - - 1.8 - 0.7
  Hemicycliophora sp. - - - 3.6 - 0.7
  Mesocriconema rusticum - - - - 3.6 0.7
  Paratylenchus sp. - - - - 1.8 0.4
  Tylenchorhynchus crassicaudatus - - - 1.8 1.8 0.7
  Tylenchus spp. 30.9 - - 25.5 18.2 14.9
 Non-parasitic nematodes
 Alaimida 1.8 - - 5.5 7.3 2.9
 Cephalobina 41.8 1.8 5.5 40.0 43.6 26.5
 Dorylaimida 30.9 - 5.5 38.2 14.5 17.8
 Monhysterida 3.6 - - 3.6 5.5 2.5
 Mononchida 3.6 - - 12.7 - 3.3
 Pangrolaimida 18.2 10.9 18.2 14.5 10.9 14.5
 Plectida - 3.6 1.8 18.2 9.1 6.5
 Rhabditida 20.0 9.1 1.8 27.3 38.2 19.3
 Triplonchida - - - 1.8 - 0.4
 Tryphylida - - 1.8 23.6 5.5 6.2
 Average 10.3 2.2 2.2 11.7 9.2 7.1
*

Total number of samples: 275

Frequency (%)=number of samples with nematodes/total number of samples examined×100.

Table 3

Nematode detected from bonsai culture media

Province City Bonsai garden No. of nematode*

Aphelen­choides Tylenchus Rhabditida Panagrolaimida Dorylaimida Mononchda Tripylida
Gyeonggi Goyang SY - - - - - - -
Gyeongnam Busan SR - - - - - - -
SB - - - 10 - - -
Changwon HR - - 20 8 - - -
GA - - - - - - -
Gyeongbuk Gyeongsan CD - - 32 - - 5 -
Pohang GR - - - - - - -
Daegu SW - - - - - - -
Jeonnam Yeosu BJ 2 10 80 - 4 - 1
Muan GS - - - - - - -
Chungnam Seosan UJ - - - - - - -
CE - - - - - - -
Daejeon HG - - - - - - -
Chungbuk Chungwon OS - - - - - - -
Okcheon SS - - - - - - -
Total 6 12 15 2 10 132 18 4 5 1
*

Number of nematode per bonsai culture medium 300 cm3

Initials of bonsai garden.