Vol. 45 No. 2
Nematological Research
December, 2015
[Original Article]
Development of real-time PCR primers specific to the garlicdamaging potato rot nematode Ditylenchus destructor to quantify its density in soil and outer skin of garlic Zejun Cheng1, Koki Toyota1,* and Kazuo Yamashita2 The potato rot nematode Ditylenchus destructor is a major threat to garlic production in Aomori Prefecture, Japan. The objectives of this study were i) to design real-time PCR primers specific to D. destructor, and ii) to make calibration curves to evaluate the relationships between the number of D. destructor inoculated to soil or outer skin of garlic and the cycle threshold (Ct) values. Ditylenchus destructor strains were collected from 11 major growing regions in Aomori Prefecture and their ITS regions were sequenced. Strains from different regions showed the same sequence and, thus, real-time PCR primers specific to D. destructor were designed. The specific primers (Ddf and Ddr) matched 100% with 10 out of 11 D. destructor sequences from different countries as well as with the Aomori strains, while there are 7 and 16 bp mismatches in the closest species D. africanus and D. askenasyi, respectively. There were highly significant correlations (soil: y = −1.1221x + 35.225, R2 = 0.9973; outer skin of garlic: y = −1.145x + 35.295, R2 = 0.9883) between the log-transformed numbers of nematodes inoculated (x) and the respective Ct values (y). Based on this calibration curve, the densities of D. destructor in soils were estimated to be 43/10 g of soil in an infested field and 0/10 g of soil in a field without a history of garlic cultivation. These results demonstrated that the presently designed primers are useful to quantify the density of D. destructor in both soil and garlic. Nematol. Res. 45(2), 93 –99 (2015). Key words: Allium sativum, Ditylenchus dipsaci, garlic bulb, primer design
INTRODUCTION Garlic (Allium sativum L.) is one of the most important flavoring agents in the world. China is the biggest garlic producing country, accounting for more s production (FAOSTAT, 2012). In than 70% of the world’ Japan, Aomori Prefecture is by far the top producer and occupies ca. 70% of the total area of garlic cultivation (MAFF, 2012). The potato rot nematode Ditylenchus destructor Thorne can cause significant damage to potato, flower bulbs and corms, and carrots (EPPO, 2008). In Japan, the nematode is known to cause serious damage to garlic since the first report in 1984 (Fujimura et al., 1986). According to a survey by Aomori Prefecture, damage s total garlic was seen in at least 10% of the prefecture’ cultivation area in 2013. Several chemical control Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan 2 Vegetable Research Institute, Aomori Prefectural Industrial Technology Research Center, Inuochise, Rokunohe, Aomori 0330071, Japan * Corresponding author, E-mail:
[email protected] 1
methods have been developed to prevent damage caused by the nematode using granular nematicides, soil fumigants such as chloropicrin, and fungicides. A small but significant part (ca. 6%) of the total population of D. destructor is found in soil at harvest of fields infested with the nematode, although the majority of D. destructor nematodes are present in damaged plants (Basson et al., 1991; Venter et al., 1993). The nematodes surviving in soil infect garlic plants in the next season and thus, soil fumigation with chloropicrin is one of the most efficient methods to disinfest soils with this nematode (Kitano and Yamashita, 2011). There is a growing concern, however, over the excessive use of chemicals, in particular fumigants. To avoid unnecessary use of fumigants, it is essential to establish reliable and rapid methods for quantifying the extent of nematode infestations, leading to a diagnosis of whether or not chloropicrin fumigation is needed. The Baermann method has been widely used to extract nematodes from various samples, including soil (Ingham, 1994). A recent study, however, reported that the Baermann method is not advisable for survey purposes (Den Nijs and Van den Berg, 2013). In addition,
─ 93 ─
第45巻 第 2 号
日本線虫学会誌
Yan et al. (2012, 2013) reported that several different commercial research organizations had different results in detecting particular nematode species. This may have been caused by unstable nematode extraction, inaccurate nematode counting, and difficulty in identifying nematodes under a microscope. To avoid such disadvantages, we have developed a real-time PCR method to directly quantify various plantparasitic nematodes in soil, e.g., Heterodera glycines Ichinohe (Goto et al., 2009), Pratylenchus penetrans Filipjev and Schuurmans Stekhoven (Sato et al., 2010), Meloidogyne incognita Kofoid and White (Min et al., 2011), and M. hapla Chitwood (Watanabe et al., 2013). Species-specific primers have been developed for D. dipsaci (Kühn) Filipjev and D. destructor (Marek et al., 2010), but those primers are not suitable for quantitative studies. The purpose of this study was to develop a real-time PCR primer set specific to the potato rot nematode D. destructor and a calibration curve to estimate its density in soil. In harvested garlic that is infested with D. destructor, most of the nematodes are distributed in the root base and outer skin of the garlic and there are few in the cloves, the edible part of garlic (Fujimura et al., 1989). Ditylenchus destructor nematodes surviving in outer skin or root base move into the cloves and multiply during storage. Thus, to measure the density of D. destructor in the outer skin at harvest is a useful means of diagnosis to predict future damage to the garlic after harvest. Therefore, a calibration curve was prepared to estimate the density of D. destructor in the outer skin of garlic. MATERIALS AND METHODS Nematodes used and DNA extraction from nematodes: Damaged garlic bulbs were obtained from 11 regions (Takko Town, Ebisawa, Kamiichikawa and Kuraishi in Gonohe Town, 2 fields in Singo Village, 2 fields in Misawa City, Rokunohe Town, Tohoku Town and Fujisaki Town) in Aomori Prefecture and nematodes were collected by slicing the garlic and then soaking the slices in distilled water. Nematode suspensions were washed one time by centrifugation (6,000 rpm, for 5 min) to remove water soluble components present in the garlic. Then, DNA was extracted from the washed nematodes (ca. 1,000 individuals) by the Plant Genomic DNA Extraction Mini Kit (FAVORGEN Biotech, Ping-Tung, Taiwan). In addition, DNA was extracted from individual nematodes by the method of Iwahori et al. (2000). A single nematode was put into a drop of water on a glass
2015年 12 月
slide, air-dried, then crushed and removed with a piece of sterilized filter paper under a microscope. The filter paper was transferred to a 200-µl Eppendorf tube and then 10 µl of lysis buffer (10 mM Tris-HCl (pH 8.0), 1 m M EDTA, 1% IGEPA L CA630 (Biomedicals, Strasbourg, France), 100 µg/ml proteinase K) was added. The macerate was frozen at −85℃ for 15 min. After thawing, the macerate was incubated at 65℃ for 1 h to s body and then at 98℃ for 15 min degrade the nematode’ to inactivate proteinase K. The macerate was used as a template for the PCR following the procedure described b y Fe r r i s e t a l . (19 93). T h e f o r w a r d -CGTAACAAGGTAAGCTGTAG-3’ ) and reverse (5’ -AGAAACACGTGCTAGGCCAAAG-3’ ) primers (5’ were used to amplify the ITS1-5.8S-ITS2 region. PCR amplification was performed with MightyAmp® DNA Polymerase Ver.2 (Takara Bio, Otsu, Japan). The reaction conditions were 94℃ for 2 min; 35 cycles of (94℃ for 1 min; 48℃ for 1 min; 72℃ for 1 min); 72℃ for 5 min. The PCR product was purified by using a FavorPrepTM GEL/ PCR Purification Mini Kit (Favorgen Biotech) and then sent to Takara Bio for sequencing. Design of specific primers: Based on the ITS regions (ca. 700 bp) of different Ditylenchus spp. (Table 1) and the D. destructor obtained in this study and from database, real-time PCR primers specific to D. destructor were designed. The designed primer set in this study showed 100% similarity among all but one of the D. destructors strains obtained (Table 2). DNA extraction from soil: Two soils (andosols) were collected from fields of the Vegetable Research Institute (Rokunohe, Aomori), Aomori Prefectural Industrial Technology Research Center: one from a field with garlic damaged by D. destructor and the other from a field without a history of garlic cultivation for at least 30 years. Collected soil samples were dried in an oven at 60℃ for 24 h, and then the samples (20 g each) were homogenized with a ball mill (MM301; Retsch, Hahn, Germany) for 2 min at a frequency of 20/s. DNA extraction was carried out by the method of Sato et al. (2010). DNA was extracted in duplicate from 0.5 g oven-dried soil and finally dissolved in 100 µl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Calibration curve for soil: As Ct values were not obtained in the non-infested soil, a calibration curve was made to estimate the relation
─ 94 ─
Vol. 45 No. 2
December, 2015
Nematological Research
Table 1. Comparison of the sequences in the positions of the specific primers to Ditylenchus destructor among D. destructor and related species. Forward primer (DdF: 5’ →3’ ) CAC GTC TGA TTC AGG GTC GTA AAT A
Forward primer (DdF: 5’ →3’ ) AGA AAC ACG TGC TAG - - - GCC AAA G
Aomori specimens
･･･ ･･･ ･･･ ･･･ ･･･ ･･･ ･･･ ･･･ ･
･･･ ･･･ ･･･ ･･･ ･･･ - - - ･･･ ･･･ ･
D. destructor (KF221214)
･･･ ･･･ ･･･ ･･･ ･･･ ･･･ ･･･ ･･･ ･
･･･ ･･･ ･･･ ･･･ ･･･ - - - ･･･ ･･･ ･
D. askenasyi (AF396337)
･ ･ ･ ･ ･ ･ ･ ･G C ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･
T ･ ･ CC ･ TA ･ ･ ･ G A ･ T AGC A ･ ･ C ･ ･ C
Target nematode (accession number)
D. dipsaci (KM008549)
･ ･ ･ A･ ･ ･ ･ G C ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･
･ A ･ ･ G ･ ･ ･ C CAA CCA GTA C ･ G C ･ T ･
D. drepanocercus (JQ429774)
･ ･ ･ A･ T ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･
T ･ ･ TTA ･ A ･ CAG ･ CA CCA CA ･ ･ C ･ C
D. gigas (HQ219240)
･ ･ ･ A･ ･ ･ ･ G C ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･
･ A ･ ･ G ･ ･ ･ C C ･ ･ ･ ･ ･ TTT ･ ･ ･ ･ TG A
D. halictus (EF627047)
･ ･ ･ ･ ･ T ･ ･ G ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ A･ ･ ･ ･ ･
･ A ･ ･ ･ ･ TAA ･ ･ ･ ･ TA - - - - T ･ T ･ ･ ･
D. gallaeformans (JQ429779)
T ･ ･ A･ ･ ･ ･ G C ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･
･ T ･ TG ･ G ･ ･ ･ T ･ ･ ･ - - - - - ･ ･ TTT T
D. africanus (KF219617)
･ ･ ･ ･ ･ ･ ･ ･G C ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･
･ ･ ･ ･ G ･ ･ A･ ･ ･ T ･ ･ ･ - - - A･ ･ ･ ･ ･ ･
Eutylenchus excretorius (EU915500)
･ ･ T A･ ･ ･ ･ G ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ C ･
TC ･ ･ ･ ･ T ･ - ･ A ･ ･ GA TCT ･ ･ ･ ･ ･ G C
Accession numbers in parentheses after the species names, hyphens indicate deletion of the corresponding base and dots indicate the same base as the primer species. Table 2. The number of mismatches in the regions of the specific primers shown in Table 1 between Aomori specimens and Ditylenchus destructor isolated from the other locations. No. of mismatch (position*) Forward Reverse primer primer
Nematode species of target sequences (accession number) China (KF221214, JX040544, FJ911551, EF418003, EF208213) Poland (GQ469492)
0
0
1(10)
0
(KC923224)
0
0
Iran (JN166693)
0
0
USA (HQ235697, AF363110, AY987007)
0
0
Canada (JX162205)
0
0
*
From the
end.
between the number of D. destructor inoculated and the Ct values. Ditylenchus destructor consisting of a mixture of juveniles, males and females (11, 56 , and 33 individuals 100/µl, respectively) was inoculated in triplicate to 10 g of the oven-dried soil at densities of 2, 10, 100, 1000 individuals. Then, DNA was extracted in duplicate from the inoculated soils and real-time PCR was done as described below. DNA extraction from garlic: Outer skin of cloves, consisting of bulb base and protection leaf, was obtained from garlics imported from Spain, which is considered not to be contaminated with the nematode, and homogenized with a grinder (FM-70e; SUN Co., Ltd., Kyoto, Japan). Homogenized outer skin samples (0.05 g) were inoculated in triplicate with different numbers of D. destructor consisting of a mixture of juveniles, males and females, and then oven-
dried at 60℃ overnight. DNA extraction from the outer skin with nematodes was carried out by two methods: one by the same soil method as described above, and the other by the method of Suga et al. (2008) which was developed to extract DNA from plant roots. In the latter method, DNA was extracted in duplicate and finally dissolved in 300 µl TE. Real-time PCR protocol: The DNA extracts from soil and outer skin of cloves were used as templates in real-time PCR after 10-fold dilution with sterile distilled water. Real-time PCR was performed using a Step One Real Time PCR System (Life Technologies, Tokyo, Japan) in final volume of 10 µl containing 5 µl of Fast SYBY Green Master Mix (Life Technologies), 0.4 µl of each primer, and 2 µl of template s recommended condition DNA under the manufacturer’ 95℃ for 30 sec; 40 cycles of (95℃ for 5 sec; 60℃ for 30
─ 95 ─
第45巻 第 2 号
日本線虫学会誌
Head
2015年 12 月
Middle
Tail
Normal
Abnormal Fig. 1. Normal and abnormal nematodes extracted from damaged garlic.
sec). A negative control was prepared using distilled water instead of a DNA template. Effect of the developmental stage of the nematode on Ct values: Nematodes obtained from damaged garlic were separated into juveniles, males, females and abnormal nematodes (Fig. 1). The abnormal nematodes were observed after a certain period of storage. They were not mobile and showed abnormality in the cells. DNA was extracted in 8 to 11 replicates from individual nematodes based on the method described above and real-time PCR was done as describe above. Statistical analysis: Statistical analysis was performed using the software Excel St atistics 2002 (Social Su r vey Research Information Co., Ltd., Tokyo, Japan). RESULTS Sequence analysis: The sequences of the ITS region of D. destructor were identical among the 11 different regions in Aomori Prefecture. Then, the sequences were compared to the related nematode species available in The National Center for Biotechnology Information (NCBI). Real-time PCR primers (DdF and DdR) were designed based on the sequences specific to D. destructor from Aomori and other countries (Table 1). The designed primers matched 100% with 10 out of 11 D. destructor sequences available in the database and only a sequence from Poland (GQ469492) showed one mismatch in the forward primer (Table 2). In contrast, the primers showed more than 7 bp mismatches to the other species (Table 1). Calibration curve for soil and detection limit: There was a highly significant negative correlation between the Ct values ( y) and the log-transformed numbers of nematodes inoculated (x) (y = −1.1221x +
35.225, R2 = 0.9973, P < 0.01) (Fig. 2). The density of D.
destructor in field soil was estimated based on the equation and the field with damaged garlic contained 43 individuals/10 g of soil, while D. destructor was not detected in soil from a field without garlic cultivation history. Ct values increased logarithmically proportionally with the dilution rate. A linear relationship between dilution rates of DNA and Ct values was detected at up to 107-fold dilution, ranging from 12.2 to 33.1 (Fig. 3), while the Ct value was not determined in 108-fold dilution and the negative control showed no Ct values. Calibration curve for garlic: Calibration curves were made using two different methods (Fig. 4). In both methods, Ct values were not always obtained at a density of 2 individuals among replicates and therefore the data were removed from the calibration curve. The slope values were comparable, but the R2 value was better in the method for root than for soil. In the root method, there was a highly significant negative correlation between the Ct values (y) and the log-transformed numbers of nematodes inoculated (x) (y = −1.145x + 35.295, R2 = 0.9883, P < 0.01). Effect of different stages on Ct values: Abnormal nematodes showed significantly higher Ct values (26.2 ± 2.0, n = 8) than normal nematodes (including females, males, and juveniles, 20.7±0.7, n = 27), indicating the loss of DNA after the abnormality. There was no significant difference in Ct values between female (19.6 ± 0.5, n = 8) and male (20.5±0.5, n = 11) nematodes, while juveniles tended to show higher Ct values (21.0±0.7, n = 8), i.e., lower copy numbers per individual, than adults, although a significant difference (P < 0.05) was observed only with females.
─ 96 ─
DISCUSSION The present st udy successf ully developed a
Vol. 45 No. 2
35
y = −1.1221x + 35.225 R2 = 0.9973
34
30
30
25
26 22
Ct value
Ct value
December, 2015
Nematological Research
20
y = −3.0356x + 12.81 R2 = 0.9943
15 0
2 4 6 8 10 Log2 (Number of D. destructor added to 10 g oven-dried soil)
12
Fig. 2. Relationship between the log-transformed numbers of Ditylenchus destructor added to 10 g of oven-dried soil and the cycle threshold (Ct) values (error bars are standard deviation; n = 4).
A
Ct value
36
y = −0.9212x + 31.985 R2 = 0.7612
32
6
7
8
9 10 11 12
-4
-3
-2
-1
0
10
y = −1.145x + 35.295 R2 = 0.9883
32
24
5
B
36
24
4
-5
Fig. 3. Effect of dilutions of a DNA template extracted from nematodes of damaged garlic on the Ct values.
28
3
-6
Log-transformed dilution rate
28
20
-7
-8
20
Log2 (Number of D. destructor added to 0.05 g oven-dried outer skin)
3
4
5
6
7
8
9
10 11
Log2 (Number of D. destructor added to 0.05 g oven-dried outer skin)
Fig. 4. Effect of two different DNA extraction methods (Sato method for soil (A) and Suga method for root (B)) on the relationship between the log-transformed numbers of Ditylenchus destructor added to 0.05 g of oven-dried outer skin of garlic and the cycle threshold (Ct) values (error bars are standard deviation; n = 4).
quantification method for D. destructor in soil and garlic. The ITS sequences determined in this study were identical among the nematodes collected from 11 different regions in Aomori Prefecture, although the genetic diversity of D. destructor in inter-sample sequence repeats is relatively high (Huang et al., 2012). In the NCBI database, different sequences are reported from different countries, Canada, China, Iran, Poland, and the USA, and all these sequences belonging to D. destructor showed the same sequences in the primer region, except one strain from Poland, in which there is only one mismatch in the 10th base pair from the 3’end (Tables 1 and 2). According to Koyama et al. (2013), one
mismatch in the 3rd position from the 3’end showed 54% amplification efficiency compared with a perfect match and Bru et al. (2008) reported that amplification efficiency is lower when a mismatch is present closer to the 3’end, suggesting that one mismatch in the 10th base pair from the 3’end might show enough amplification efficiency. In contrast, the amplification efficiency becomes less than 0.6% when there are more than 2 mismatches in the regions of 10 base pairs from the 3’end of the primer (Kawanobe et al., 2015). The nearest species D. africanus showed 2 mismatches in the region and a total of 7 mismatches, suggesting that even the nearest species shows very low amplification efficiency
─ 97 ─
第45巻 第 2 号
日本線虫学会誌
to the primer set (DdF and DdR) specif ic to D. destructor. These results suggest that the primer set designed in this study specifically amplifies a sequence present in D. destructor throughout the world as well as in different regions in Aomori Prefecture, the major garlic producing prefecture in Japan. To test the performance of this primer set, soil was collected from fields with two different histories: one field with garlic damaged by D. destructor and the other without a history of garlic cultivation. The former soil showed a large number (43/10 g soil) of nematodes, while the latter had zero, suggesting that the density of D. destructor in soil was related to the garlic plant damage caused by D. destructor. As a future study, evaluation of the relationship between the pre-planting density of D. destructor in soil and damage to garlic is planned. A calibration curve was made to estimate the density of D. destructor in the outer skin of garlic cloves (Fig. 4). To extract DNA from garlic, the same method was used as for soil, but variations among replicates were large and the correlation coefficient values were low. Thus, another method, which has been reported to extract DNA from root samples, was used. The root method provided a reliable calibration curve, enabling the quantification of the nematodes in a small amount (0.05 g) of outer skin, which has no commercial value. The results show that the root method is better than the soil method for the outer skin samples. We need to pay attention to quantification at low densities. Ct values (33 . 9±1.5 and 31.8±1. 0) were obtained in all the replicates when 2 and 10 individuals were inoculated to 10 g soil and 0.05 g of outer skin of garlic, respectively (Figs. 2 and 4). However, Ct values were not always observed at densities of 1/10 g of soil in our previous experiments and 2/0.05 g of outer skin in this study (data not shown). Thus, the minimum detection limit was considered to be 2/10 g of soil and 10/0.05 g of outer skin. When a positive sample was serially diluted, a logarithmically linear relation in the Ct values was observed between 12.1 and 33.1 and no Ct value was detected in further diluted samples (Fig. 3). Thus, caution is necessary to interpret Ct values higher than 33.1, corresponding to less than 3.7/10 g of soil based on the equation in Fig. 2 and less than 3.8/0.05 g of outer skin based on the equation in Fig. 4. In such cases, a Ct value cannot be obtained even if the target nematode is present. Accuracy at low densities remains as a challenge for future study. Contradicting results have been reported for differences in Ct values for the various life stages. Yan et
2015年 12 月
al. (2013) and Kawanobe et al. (2015) reported that there were no significant differences in the Ct values among juveniles and adults of Pratylenchus neglectus Rensch and P. penetrans, respectively. In contrast, this study and Sato et al. (2007) found that adults had lower Ct values, i.e., higher copy numbers of DNA, than juveniles. An exact reason for this difference remains to be solved. In this study, D. destructor nematodes were extracted from diseased garlic. Among the nematodes extracted, a small part of the nematodes were not mobile and had dark colored bodies. It is difficult to conclude whether or not such nematodes are dead, but their metabolic activity must be quite low, as they were immobile. Such nonmobile nematodes showed Ct values 5 cycles higher, i.e. 32 times lower copy numbers, than juveniles, suggesting that the quantity of DNA in living nematodes decreases rapidly after they lose their activity. ACKNOWLEDGEMENTS We would like to express our thanks to Mr. A. Kushida, Hokkaido Agricultural Research Center, for his useful suggestions, Ms. N. Yamashita, Sannohe Agricultural Extension Office and garlic farmers for their assistance in sampling, and Ms. N. Sasaki and Ms. S. Ohshita, Aomori Prefectural Industrial Technology Research Center, for their technical assistance. LITERATURE CITED Basson, S., De Waele, D. G. M. A. and Meyer, A. J. ( 1991) Population dy namics of Dit ylenchus destructor on peanut. Journal of Nematology 23, 485 –490. Bru, D., Martin-Laurent, F. and Philippot, L. (2008) Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Applied and Environmental Microbiology 74, 1660 –1663. Den Nijs, L. and Van den Berg, W. (2013) The added value of proficiency tests: choosing the proper method for extracting Meloidogyne second-stage juveniles from soils. Nematology 15, 143–151. EPPO (2008) Ditylenchus destructor and Ditylenchus dipsaci. EPPO Bulletin 38, 363–373. FAOSTAT (2012) ht t p://faostat.fao.org /site/ 567/ desktopdefault.aspx#ancor Ferris, V. R., Ferris, J. M. and Faghihi, J. (1993) Variation in spacer ribosomal DNA in some cyst-forming species of plant parasitic nematodes. Fundamental and Applied Nematology 16, 177–184. Fujimu ra, T., Ichida, T. and K imu ra, T. (1989)
─ 98 ─
Vol. 45 No. 2
Nematological Research
Occurrence of potato-rot nematode, Ditylenchus destructor Thor ne, in garlic and cont rol. 1. Evaluation of treatments applied before planting and after harvest for control. Japanese Journal of Nematology 18, 22–29. (in Japanese with English summary) Fujimura, T., Washio, S. and Nishizawa, T. (1986) Garlic a s a new host of t he pot ato -rot nematode, Ditylenchus destructor Thorne. Japanese Journal of Nematology 16, 38 –47. (in Japanese with English summary) Goto, K., Sato, E. and Toyota, K. (2009). A novel detection method for the soybean cyst nematode Heterodera glycines using soil compaction and realtime PCR. Nematological Research 39, 1–7. Huang, W., He, X., Ding, Z. and Peng, D. (2012) Genetic var iation of Dit ylenchus destr uctor and its association with ecological factors. Journal of Agricultural Biotechnology 20, 62– 69. (in Chinese with English summary) Ingham, R. E. (1994) Nematodes. In: Methods of soil analysis. Part 2. Microbiological and biochemical properties (Weaver, R. W. ed.), American Society of Agronomy, Madison, 459–490. Iwahori, H., Kanzaki, N. and Futai, K. (2000) A simple, polymerase chain reaction-restriction fragment length polymorphism-aided diagnosis method for pine wilt disease. Forest Pathology 30, 157–164. Kawanobe, M., Miyamaru, N., Yoshida, K., Kawanaka, T. and Toyota, K. (2015) Quantification of lesion nematode (Pratylenchus zeae), stunt nematode (Tylenchorhynchus leviterminalis), spiral nematode (Helicotylenchus dihystera) and lance nematode (Hoplolaimus columbus), parasites of sugarcane in Kitadaito, Okinawa, Japan, using real-time PCR. Nematological Research, in press. Kitano, N. and Yamashita, K. (2011) Effect of soil fumigant on damage of garlic caused by Ditylenchus destructor. Annual Report of the Society of Plant Protection of North Japan 62, 144–147 (in Japanese) Koyama, Y., Thar, S. P., Kizaki, C., Toyota, K., Sawada, E. and Abe, N. (2013) Development of specific primers to Hirschmanniella spp. causing damage to lot us and their economic th reshold level in Tokushima Prefecture in Japan. Nematology 15, 851– 858. MAFF (2012) http://www.maff.go.jp/tohoku/nouson/ kokuei/kitaouu/syoukai.html Marek, M., Zouhar, M., Douda, O., Mazakova, J. and Rysanek, P. (2010) Bioi n for mat ics-assisted
December, 2015
characterization of the ITS1-5.8S-ITS2 segments of nuclear rRNA gene clusters, and its exploitation in molecular diagnostics of European crop-parasitic nematodes of the genus Ditylenchus. Plant Pathology 59, 931–943. Min, Y. Y., Toyota, K., Goto, K., Sato, E., Mizuguchi, S., Ab e , N., Na k a n o, A ., a n d S a t o, E . ( 2011) Development of a direct quantitative detection method for Meloidogyne incognita in sandy soils and its application to sweet potato cultivated fields in Tokushima Prefecture, Japan. Nematology 13, 95 –102. Sato, E., Goto, K., Min, Y. Y., Toyota, K. and Suzuki, C. (2010) Quantitative detection of Pratylenchus penetrans from soil using soil compaction and realtime PCR. Nematological Research 40, 1– 6. Sato, E., Min, Y. Y., Shirakashi, T., Wada, S. and Toyota, K. (2007) Detection of the root-lesion nematode, Pratylenchus penetrans (Cobb), in a nematode community using real-time PCR. Japanese Journal of Nematology 37, 87–92. Suga, Y., Hori, K., Komori, S., Fukunaga, A., Ikeda, J. and Toyota, K. (2008) Simple and rapid DNA extraction method from plant roots for analysis of bacterial community structure. Soil Microorganisms 62, 121–125. (in Japanese) Venter, C., De Waele, D. and Meyer, A. J. (1993) Reproductive and damage potential of Ditylenchus destructor on six peanut cultivars. Journal of Nematology 25, 59 – 62. Watanabe, T., Masumura, H., Kioka, Y., Noguchi, K., Min, Y. Y., Murakami, R. and Toyota, K. (2013) Development of a direct quantitative detection method for Meloidogyne incognita and M. hapla in andosol and analysis of relationship between the initial population of Meloidogyne spp. and yield of eggplant in an andosol. Nematological Research 43, 21–29. Yan, G., Smiley, R. W. and Okubara, P. A. (2013) Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Disease 97, 757–764. Yan, G., Smiley, R. W., Okubara P. A., Skantar, A. M. and Reardon, C. L. (2012) Detection and quantification of Pratylenchus thornei in DNA ex t r a c t e d f rom soi l u si ng r e a l-t i me PC R . Phytopathology 102, 14–22.
─ 99 ─
Received: 2 February, 2015
