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ExpansinB3 as a marker for detecting pine wood nematode-infected pine trees
Accepted Manuscript ExpansinB3 as a marker for detecting pine wood nematodeinfected pine trees
Young Ha Kim, A-Young Kim, Bo-Hye Choi, Hye-Rim Han, Young Ho Koh PII: DOI: Reference:
S1226-8615(17)30437-5 doi: 10.1016/j.aspen.2017.08.029 ASPEN 1058
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
19 July 2017 25 August 2017 30 August 2017
Please cite this article as: Young Ha Kim, A-Young Kim, Bo-Hye Choi, Hye-Rim Han, Young Ho Koh , ExpansinB3 as a marker for detecting pine wood nematode-infected pine trees, Journal of Asia-Pacific Entomology (2017), doi: 10.1016/j.aspen.2017.08.029
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ExpansinB3 as a marker for detecting pine wood nematode-infected pine trees
1, Department of Biomedical Gerontology, Graduate School of Hallym University, Chuncheon,
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Young Ha Kim1,2,*, A-Young Kim1,2,*, Bo-Hye Choi1,2, Hye-Rim Han3, and Young Ho Koh1,2
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Gangwon-do, Republic of Korea
Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Republic of Korea
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Laboratory of Pine Wilt Disease, Division of Forest Insect Pests & Disease, National Institute
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2.
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of Forest Science, Seoul, Korea Short title: EXPB3, a marker for pine wilt disease
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* These authors contributed equally.
P Correspondence to Dr. Hye Rim Han email: [email protected] and Dr. Young Ho Koh email:
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[email protected]
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Abstract
To develop a method for recognizing pine trees infected with pine wood nematodes (PWN),
Bursaphelenchus xylophilus, we have constructed a monoclonal antibody (Mab) library for We conducted
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Expansin B3 (EXPB3), which is one of the known proteins that is secreted by PWN.
A secondary screening was performed using the PBS extracts of the PWN-
infected pine trees (PWNIPTs).
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immunoreactivity.
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a primary screening by using the EXPB3 peptides and selected 96 fusion cell lines with a high
Thirteen clones, including 9 clones with high immunoreactivities,
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were selected, and limited dilutions were performed to establish Mab-secreting hybridoma lines.
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A total of 74 PWN infected pine trees collected from 9 PWN-affected areas in the Republic of Korea were analyzed by using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4).
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recognized all of the infected pine trees with some variations.
EXPB3-C11-E4
This Mab demonstrated a
significantly higher reactivity to the black pines when compared to the red pines.
This Mab
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recognized both PWN-infected red and black pine trees collected from different regions, but were
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able to displayed regional differences with the black pine trees.
In addition, the EXPB3-C11-E4
immunoreactivity in black pine trees had a significant correlation regarding the degree of PWN infection and the perimeters of the trees.
The results of this study reveal that EXPB3, an antigen
secreted by PWN, and its specific Mabs may be used for the diagnosis of PWNIPTs.
Key word: EXPB3, Mab library, red pine trees, black pine trees, PWN infected pine tree
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Introduction
Pine wilt disease (PWD) is currently the most devastating coniferous forest disease in the world and has been proven to be caused by an infection from the pine wood nematode (PWN),
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Bursaphelenchus xylophilus (Steiner & Buhner, 1934) Nickle (Futai, 2013). Since first reported in
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Nagasaki, Japan in 1905, this disease has spread to other East Asian countries, including China in In addition, PWN infected
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1982, Taiwan in 1985, and Korea in 1988 (Futai, 2013; Suzuki, 2002).
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pine trees were observed in the Iberian Peninsula, Portugal in 1999 (Futai, 2013; Mota et al., 1999) and then were observed in the Cáceres Region of Spain near Portugal in 2008 (Abelleira et al.,
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2011). The disbursement of the PWN within a local area is mediated by several pine sawyer However, it is known that the occurrence of PWD
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beetle species in the genus Monochamus spp.
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in remote areas or other continents are caused by an artificial transport of the PWN and its vector infected trees (Futai, 2013). Therefore, the best way to currently prevent a widespread outbreak
Concurrently, locating and burning the PWN infected trees is one of the most
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uninfected areas.
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of PWD is to prevent the transportation of pine trees from the PWN-infected areas to other
effective ways to prevent the spread of the PWD since the elimination of infected trees reduces the density of the vectors (Futai, 2013).
Various research efforts have been conducted in order to detect and confirm the PWN and its infected pine trees.
Prior to developing diagnostic methods using molecular markers,
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confirmation of the PWN infection of pine trees was based on the microscopic observation of extracted nematodes in pine trees (Futai, 2013; Yik and Birchfield, 1981). Diverse molecular markers including mitochondrial genes (Cheng et al., 2008; Kanzaki and Futai, 2002), internal
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transcribed regions (Cheng et al., 2008; Kanzaki and Futai, 2002), and various micro-satellites (Jung et al., 2010; Mallez et al., 2013) were identified and used to develop techniques.
However, most
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of the molecular tools required sophisticated machines and sometimes it was necessary to separate PCR products on agarose gels to finish a diagnosis.
To overcome these limitations,
However, the LAMP reaction mixtures must be incubated
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(Kang et al., 2015; Kikuchi et al., 2009).
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several methods using loop-mediated isothermal amplification (LAMP) have been developed
for at least one h at approximately 60°C, and strong fluorescence signals must be confirmed by
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UV illumination or other methods, in addition to requirements of extractioin genomic DNA from
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wood pellets.
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To overcome those limits, rapid testing kits using antigen-antibody interaction for quick In a previous study, we reported that Expansin B3
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diagnosis in the field must be developed.
(EXPB3) is a protein secreted from PWNs when they are infecting pine trees (Lee et al., 2012).
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this study, a monoclonal antibody (Mab) secreting fusion hybridoma library for EXPB3 was constructed and Mabs selectively recognizing PWN-infected pine trees (PWNIPTs) were screened. In addition, specificities and sensitivities of Mabs were confirmed by using various PWNIPTs collected from nine regions in Korea. 4
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Material and Methods
The target protein identification and synthesis of peptides for antigens
In previous study, we reported that ExpansinB3 (EXPB3) was highly expressed in
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propagative and dispersive stages in PWN (Lee et al., 2012). The amino acid sequence of EXPB3
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was used to blast using a B. xylophilus database in GeneDB
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(http://www.genedb.org/Homepage/Bxylophilus) and NCBI Blast
Based on the blast results, two
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(https://blast.ncbi.nlm.nih.gov/Blast.cgi) (data not shown).
PWN and other nematode species.
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peptides were designed showing amino acid sequence discrepancy to various EXP homologues in The sequences of the two synthesized peptides were 42The two peptides were
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CGLDIANLSAGVSSKLFD-59 and 141-KPATITYLKCSDTTPTTNC-150.
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conjugated with a keyhole limpet hemocyanin (KLH) and then used as antigens for immunizing
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four Balb/C mice (Peptron, Daejeon, Korea).
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Immunization and primary screening of EXPB3 specific monoclonal antibodies
The fusion hybridoma cell line constructing experiment was approved by experimental animal ethic committees at Hallym University (No. HMC2016-00622-7). We followed the standard monoclonal antibody (Mab) generation protocol that was used in Lee et al. (Lee et al., 2011). supernatants of fusion hybridoma recognizing two peptide antigens were screened by using 5
The
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enzyme linked immunosorbent assay (ELISA). Antigenic peptides (0.1 g/ml) were used to coat 96-well plates, which were then blocked with 5.0% non-fat-dried milk in PBS for one h at room temperate (RT) with shaking. After three wash cycles with 0.1% Triton X-100 in PBS (PBST), the
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supernatants of the 1920 fusion hybridoma cell lines were added and incubated at four °C overnight. After three wash cycles with PBST, the peroxidase conjugated anti-mouse IgG, A, and
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M antibodies (ThermoFisher Scientific, Waltham, MA, USA) were applied and incubated at RT for two h. One step-Ultra TMB solutions (ThermoFisher Scientific) were used to detect the
Optical densities (ODs) in each well were measured at 450 nm.
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Winooski, VT, USA).
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peroxidase activities which were then measured by using an Epoch ELISA plate reader (Biotek,
Wood pellets from 74 PWNIPT and 22 healthy pine trees (HPT) were collected by using a
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wooden pellet collector (FJ Technology Co., LTD, Seoul., Korea) from nine regions or three regions, respectively, in Korea (Supplementary Table 1). Collected wooden pellets were immediately
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transferred to the laboratory and then kept in refrigeration at four °C until used.
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To determine the optimal extraction solution, we used dH2O alone or with 0.1% Triton X100 (H2OT) and phosphate buffered saline (PBS, 0.01 M sodium phosphate, 0.154 M NaCl, pH 7.2) alone or with 0.1% Triton X-100 (PBST).
A total of five ml of four different extraction solutions
were mixed with 0.5 g of wooden pellets and incubated for five min at RT, and the solutions were then carefully decanted to avoid debris. Among the solutions, the PBS extracts displayed higher
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immunoreactivities to selected supernatants (Fig. 1).
Therefore, all other experiments were
performed using PBS as an extracting solution in this study.
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Secondary screening with the pooled PBS extracts of 74 PWN infected pine trees
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The selected 96 hybridoma lines were further screened by performing ELISA as described This screening
Thirteen clones with high ODs were selected for single cell cloning.
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Single cell cloning by limited dilution
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was done in three iterations.
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above except for using the pooled PBS extracts of 74 PWNIPT as immunogens.
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Limited dilution was performed as previously described to obtain single cell clones of 13 hybridoma lines showing a higher OD to pooled PWNIPT (Lee et al., 2011).
The supernatants The
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from the limited diluted clones were screened using pooled PBS extracts of the 74 PWNIPT.
as controls.
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PBS extracts from 22 healthy pine trees (HPT) from three regions in Korea were pooled and used The ODs of each Mab to the pooled PBS extracts of 74 PWNIPT were normalized by
the ODs of the same Mab to the pooled PBS extracts of 22 HPT.
The equation is as follows:
Normalized specificity = (an OD of the pooled PBS extracts of 74 PWNIPTs – an OD of the pooled PBS extracts of 22 HPTs)/an OD of the pooled PBS extracts of 22 HPTs.
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Measuring the normalized specificity of EXPB3-Mab-3-1-H3-C11 to 74 PWNIPTs
PBS extracts of 74 PWNIPTs from nine different regions in Korea were used since there
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may be unknown variations among PWNIPTs according to the regions and stages of infections. The
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The hybridoma cell culture medium was used for the control and background signals.
Wood pellets from 42 red pine trees and 32 black pine trees were used in this study
(Supplementary Table 1).
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screenings.
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selected hybridoma line, EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) was used for further
The degree of progression of the PWD in pine trees used in the study
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ranged from normal (zero) to completely dead (six) by visual inspections and presence of the
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PWN. Additionally, the perimeter of infected trees was measured at 1.2 m above ground and
Statistical analysis
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was used for correlation analyses (Supplementary Table 1).
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The differences between the groups were analyzed by using Analysis of Variance (ANOVA) with the Excel program (Microsoft, Redmond, WA, USA).
Tukey-Kramer post hoc analysis was
performed in order to determine differences among groups by using the Excel program (Microsoft). The Pearson correlation coefficient among ODs, degree of disease progress, and the perimeter of trees at 1.2 m above ground were calculated by using the Excel program (Microsoft). 8
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If Pearson correlation coefficient is 0.1 to 0.2 or -0.1 to -0.2, the correlation is considered low.
If
the Pearson correlation coefficient is 0.2 to 0.4 or -0.2 to -0.4, the correlation is considered medium.
If Pearson correlation coefficient is 0.4 to 1.0 or -0.4 to -1.0, the correlation is
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considered high.
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Results
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Primary screening using antigenic peptides identified 96 hybridoma lines secreting EXPB3
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antibodies
The EXPB3 monoclonal antibody (Mab) library containing 1,920 fusion hybridoma lines
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was generated for screening Mabs that recognized PWNIPTs. The primary screening was performed to identify fusion hybridoma lines secreting EXPB3 peptides recognizing Mabs which We normalized the ODs of each 96-well plate by using the
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were used as antigens by ELISA.
highest OD in each plate because there were some variations among the plates. The cut-off
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value of normalized OD obtained from ELISA was > 40% of the highest OD for each plate. After primary screenings, 96 fusion cells were selected for further study (Supplementary Fig. 1).
PBS was a suitable solution for extracting antigens for EXPB3-Mabs
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Selecting solutions that can most efficiently extract the antigens is one of the important steps for the development of a diagnostic technology.
Particularly, it is important to select an
extraction solution that does not contain health or environmental hazard chemicals because the
harmful to humans or the environment.
Therefore, we choose dH2O and PBS with or without 0.1%
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The LD50 of Triton X-100 is 1,800 mg/kg when administered orally to rats (MSDS,
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Triton X-100.
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diagnosis method used in the field could be problematic if the solutions contain substances
exposure to humans and other wild animals.
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2013). Thus, an addition of 0.1% Triton X-100 to solutions would not cause a severe toxic
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Four EXPB3-Mab3-1-H3-C11 sub-clones were used for performing ELISA with 4 different extracts to compare the extraction efficiency of the four solvents (Fig. 1). Among the four EXPB3-
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Mabs, EXPB3-Mab-3-1-H3-C11-E3 and –E4 had higher ODs when compared with two other Mabs. Although the ODs were different among the four Mabs, they displayed similar OD trends to the PBS extracts from PWNIPTs from Pyeoson, Jeju-do
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four extraction solutions and three PWNIPT.
or PBST.
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and Iksan, Jellabuk-do displayed relatively high ODs when compared with extracts with H2O, H2OT, However, dH2O extracts from a PWNIPT from Uiryeong, Gyeongsangnam-do displayed
higher ODs than other extracts (Fig. 1).
These results suggest that PBS may be suitable for
extracting antigens from PWNIPTs.
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Nine hybridoma lines showed specificity to PBS extracts of PWN-infected pine trees
The PBS extracts of PWNIPTs were used to analyze specificity of each hybridoma by ELISA in order to identify hybridoma lines secreting Mabs capable of recognizing PWNIPTs among the We obtained 74 PWNIPTs containing 42 red pine trees and 32 black
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96 selected hybridoma lines.
The OD of the 96 hybridoma lines
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74 PWNITs were pooled and used for secondary screening.
The PBS extracts from the
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pine trees from nine PWN affected regions (Supplementary Table 1).
were normalized by using EXPB3-Mab-3-3-D9, which has the highest OD. Nine hybridoma lines
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revealed a normalized OD value above 0.2 (Fig. 2).
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Single clones from EXPB3-Mab-3-3-D9 and -3-3-H3 had high normalized specificity to
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PWNIPTs
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To established single cell clones, cells in 13 lines including nine lines with OD value > 0.2 had limited dilution on 96 well plates.
The supernatants from the 96 wells were screened by
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using pooled PBS extracts of 74 PWNIPTs and 22 HPTs to calculate normalized specificities. Among 13 lines examined, two lines, EXPB3-Mab-3-1-C9 and -3-2-G11, failed to divide and could not execute ELISA. Single cell clones from five hybridoma lines including EXPB3-Mab-3-2-E1, EXPB3-Mab3-2-G9, EXPB3-Mab-3-2-F1, EXPB3-Mab-3-2-E1, and EXPB3-Mab-3-1-G4 displayed very low normalized specificities (< 1.0) (Supplementary Fig. 2E–I). 11
Single cell clones from EXPB3-
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Mab-3-1-C3, EXPB3-Mab-3-1-F4, EXPB3-Mab-3-1-A5, and EXPB3-Mab-3-2-E9 displayed medium normalized specificities ranging between 1.04~2.46 (Supplementary Fig. 2A-D).
Single clones from two lines, EXPB3-Mab-3-3-D9 (EXPB3-D9) and EXPB3-Mab-3-1-H3
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(EXPB1-H3) had a higher normalized specificity than those from other lines (Fig. 3).
Three of the Those
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single cell clones of EXPB3-D9 and EXPB1-H3 had a normalized specificity of 3.4 or more.
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clones were used for further studies.
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Immunoreactivity variations in PWNIPT revealed by EXPB3 antibodies
The extent of the disease progression in PWNIPTs used in this study was determined only Therefore, there is no
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by the external characteristics of the trees at the time of pellet harvesting.
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information regarding the detailed infection period or physiological change for each pine tree.
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We used the developed EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) to measure the amount of antigens present in each of the 74 PWNIPTs collected from the nine different locations (Fig. 4A).
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We compared the ODs of EXPB3-C11-E4 for the red pine trees with those of the black pine trees and found that the ODs of the black pine trees were statistically significantly higher than those of the red pine trees (Fig. 4B).
Black pine trees collected from three regions in Jejudo
tended to have higher ODs than those of the mainland region when comparing 32 black pine trees collected from five regions in Korea (Fig. 4C). 12
However, there was no statistically significant
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difference among the 42 red pine trees collected from seven regions in Korea (Fig. 4D).
There was low correlation between the ODs and degree of disease progression for the red pine trees (Pearson correlation coefficient = -0.174). However, there was a medium degree of
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correlation between the perimeter of the trees at 1.2 meters above ground and the degree of
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disease progression as well as between the perimeter of the trees at 1.2 meters above the ground In contrast,
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and the ODs (Pearson correlation coefficients: -0.21529 and 0.34562, respectively).
there was high correlation between ODs and the degree of disease progression as well as
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between ODs and the perimeter of the trees at 1.2 m above ground (Pearson correlation However, there was medium
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coefficients: 0.49583 and 0.43023, respectively) for black pine trees.
correlation between the degree of disease progression and the perimeter of the trees at 1.2 m The results of this
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above ground (Pearson correlation coefficient: -0.21069) for black pine trees.
analysis suggested that there is a difference in EXBP3-Mab reactivity depending on the species,
Discussion
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distribution area and developmental stage for individual black pine trees.
The major goals of this study were to generate an array of EXPB3-Mabs that can be used for the rapid diagnosis of PWNIPTs in the field and establish a method for collecting wooden pellets in order to complete an accurate diagnosis. 13
To accomplish this goal, we first identified
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the suitable solutions that can extract the antigens most efficiently from the pellet of the PWNIPTs. We used the PBS extracts of the PWNIPTs to identify the most specific Mabs.
EXPB3-Mabs were
screened using not only for the high reactivity to the PBS extracts of PWNIPTs but also for the low
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reactivity to the PBS extracts of HPTs to increase the accuracy and sensitivity of EXPB3-Mabs. Therefore, the EXPB3-Mabs were selected based on a normalized sensitivity calculated with the
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Methods section methodology might have high specificity and sensitivity to the PWNIPTs.
Since other research papers report on methods for detecting a PWN from the PWNIPTs,
We considered the physical characteristics of the Korean adults, and the
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Schröder et al., 2009).
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pellets of PWNIPTs collected from trunks at 1.3 m above ground (Magnusson et al., 2007;
sampling position was determined to be located at 1.2 m above ground.
This methodology was
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applied to all the samples similarly to increase the possibility of application in the field.
Our research concluded that the black pine trees among the examined PWNIPTs showed
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statistically significant higher reactivity to EXPB3-Mabs than the red pine trees (Fig. 4B).
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Additionally, the reactivity for the black pine trees displayed a significant correlation with the degree of disease progression and the perimeters of the trees.
This result suggests that, in
contrast to red pine trees, the PWD progression in black pine trees can be predicted by the reactivity to the EXPB3-C11-E4 antibody.
EXP is an enzyme present in plants and has a significant role in mediating cell wall
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loosening, and it reduces the adhesion between adjacent cell wall polysaccharides to enable normal development (Marowa et al., 2016).
In addition, the EXPs of the plant parasitic
nematodes have a function similar to the EXPs of the plants, and therefore, they might assist the
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invasion of nematodes into the host plants (Qin et al., 2004). The EXP3-Mabs developed in this study will be an invaluable resource not only for a developing rapid detection tool, but for also
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studying the mechanisms underlying the invasion and propagation of PWN in pine trees.
Although the various genetic makers that can identify PWNs have been reported (Cheng
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et al., 2008; Futai, 2013; Jung et al., 2010; Kanzaki and Futai, 2002; Mallez et al., 2013), protein
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markers for detecting PWNIPTs were not reported before our investigation. Recently, the secreted proteins from B. xylophilus when they were cultured in pine wood extracts were reported
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(Shinya et al., 2013). However, no study has been done to determine whether these secreted proteins are actually present in PWNIPTs. Since our data provided strong evidence that EXPB3
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was present in PWNIPTs, EXPB3 is the first protein biomarker for detecting PWNPITs.
Further
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researches in developing rapid diagnosis kit will allow us to do on-site diagnosis which can be completed within several min.
Acknowledgement 15
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This work was supported by a research grants from National Institute of Forest Science (Title: Development of Bursaphelenchus xylophilus detection techniques by using its specific antigens No. 12178-0279-01).
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and antibodies.
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Figure legends
Fig. 1. EXPB3-Mabs displayed a relatively high immunoreactivity to PBS extracts. Compared with H2O, H2O with 1.0% Triton X-100 (H2OT), or PBS with 1.0% Triton X-100 (PBST) extracts, PBS The PBS extracts from two PWNIPTs obtained from
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extracts revealed high optical densities (ODs).
However, the H 2O
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Pyeoson, Jeju-do and Iksan, Jellabuk-do had higher ODs than other extracts.
The immunoreactivity of 96 selected hybridoma to PBS extracts from PWNIPTs.
PBS
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Fig. 2.
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extracts from a PWNIPT from Uiryeong, Gyeongsangnam-do had higher ODs.
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extracts from 74 PWNIPTs collected from 9 regions of Korea were pooled and used.
Fig. 3. Limited dilution results of two EXPB3 hybridoma clones.
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To select single clones from 13 EXPB3 hybridoma lines, limited dilutions were performed. (A) The
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normalized specificities of 60 supernatants from the limited diluted cells from EXPB3-Mab-3-1-D9. (B) The normalized specificities of 48 supernatants from the limited diluted cells from EXPB3-Mab3-1-H3.
Fig. 4.
Three clones from each line were selected for further analysis.
Immunoreactivity of the EXPB3-C11-E4 to PWNIPTs. 17
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(A) The map of Korea showing nine locations where 74 PWNIPTs were collected. (B) Comparison of
immunoreactivity
between
PWN-infected
red
and
black
pines.
(C)
Comparison
of
immunoreactivity to PWN-infected black pines among different collection sites. (D) Comparison of
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immunoreactivity to PWN-infected red pines among different collection sites.
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Fig. 1.
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Fig. 2. 20
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Fig. 3.
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Fig. 4.
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Mallez, S., Castagnone, C., Espada, M., Vieira, P., Eisenback, J.D., Mota, M., Guillemaud, T., Castagnone-Sereno, P., 2013. First Insights into the genetic diversity of the pinewood nematode in its native area using new polymorphic msicrosatellite loci. PLoS One 8, e59165. Marowa, P., Ding, A., Kong, Y., 2016. Expansins: roles in plant growth and potential applications in crop improvement. Plant Cell Rep. 35, 949-965. Mota, M.M., Braasch, H., Bravo, M.A., Penas, A.C., Burgermeister, W., Metge, K., Sousa, E., 1999. First report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology 1, 727-734. 23
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MSDS, 2013. Material Safety Data Sheet: Triton X-100. DaeJeong Chemicals & Metals, pp. 1-8. Qin, L., Kudla, U., Roze, E.H., Goverse, A., Popeijus, H., Nieuwland, J., Overmars, H., Jones, J.T., Schots, A., Smant, G., Bakker, J., Helder, J., 2004. Plant degradation: a nematode expansin acting on plants. Nature 427, 30-30. Schröder, T., McNamara, D.G., Gaar, V., 2009. Guidance on sampling to detect pine wood nematode
Bursaphelenchus xylophilus in trees, wood and insects. EPPO Bulletin 39, 179-188. Shinya, R., Morisaka, H., Kikuchi, T., Takeuchi, Y., Ueda, M., Futai, K., 2013. Secretome analysis of the
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pine wood nematode Bursaphelenchus xylophilus reveals the tangled roots of parasitism and its potential for molecular mimicry. PLoS One 8, e67377.
Suzuki, K., 2002. Pine wilt disease – a threat to pine forest in Europe. Dendrobiology 48, 71-74.
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Yik, C.-P., Birchfield, W., 1981. Observations on the morphology of the pine wood nematode,
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Bursaphelenchus xylophilus. J. Nematol. 13, 376-384.
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Graphical abstract
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Highlights 1.
EXPB3 specific Mabs were screened from 1920 clones
2.
Among them, 13 clones were limited diluted to select single clones
3.
EXPB3-1-H3 clones showed highest specificity to pine wood nematode infected pine trees (PWNIPT) There were differences between red pine trees and black pine trees in EXPB3 Mab
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4.
immuno-reactivity.
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These EXPB3 Mab could be used to detect PWNIPTs at the field
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5.
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Young Ha Kim, A-Young Kim, Bo-Hye Choi, Hye-Rim Han, Young Ho Koh PII: DOI: Reference:
S1226-8615(17)30437-5 doi: 10.1016/j.aspen.2017.08.029 ASPEN 1058
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
19 July 2017 25 August 2017 30 August 2017
Please cite this article as: Young Ha Kim, A-Young Kim, Bo-Hye Choi, Hye-Rim Han, Young Ho Koh , ExpansinB3 as a marker for detecting pine wood nematode-infected pine trees, Journal of Asia-Pacific Entomology (2017), doi: 10.1016/j.aspen.2017.08.029
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ExpansinB3 as a marker for detecting pine wood nematode-infected pine trees
1, Department of Biomedical Gerontology, Graduate School of Hallym University, Chuncheon,
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Young Ha Kim1,2,*, A-Young Kim1,2,*, Bo-Hye Choi1,2, Hye-Rim Han3, and Young Ho Koh1,2
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Gangwon-do, Republic of Korea
Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Republic of Korea
3.
Laboratory of Pine Wilt Disease, Division of Forest Insect Pests & Disease, National Institute
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2.
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of Forest Science, Seoul, Korea Short title: EXPB3, a marker for pine wilt disease
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* These authors contributed equally.
P Correspondence to Dr. Hye Rim Han email: [email protected] and Dr. Young Ho Koh email:
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[email protected]
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Abstract
To develop a method for recognizing pine trees infected with pine wood nematodes (PWN),
Bursaphelenchus xylophilus, we have constructed a monoclonal antibody (Mab) library for We conducted
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Expansin B3 (EXPB3), which is one of the known proteins that is secreted by PWN.
A secondary screening was performed using the PBS extracts of the PWN-
infected pine trees (PWNIPTs).
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immunoreactivity.
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a primary screening by using the EXPB3 peptides and selected 96 fusion cell lines with a high
Thirteen clones, including 9 clones with high immunoreactivities,
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were selected, and limited dilutions were performed to establish Mab-secreting hybridoma lines.
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A total of 74 PWN infected pine trees collected from 9 PWN-affected areas in the Republic of Korea were analyzed by using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4).
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recognized all of the infected pine trees with some variations.
EXPB3-C11-E4
This Mab demonstrated a
significantly higher reactivity to the black pines when compared to the red pines.
This Mab
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recognized both PWN-infected red and black pine trees collected from different regions, but were
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able to displayed regional differences with the black pine trees.
In addition, the EXPB3-C11-E4
immunoreactivity in black pine trees had a significant correlation regarding the degree of PWN infection and the perimeters of the trees.
The results of this study reveal that EXPB3, an antigen
secreted by PWN, and its specific Mabs may be used for the diagnosis of PWNIPTs.
Key word: EXPB3, Mab library, red pine trees, black pine trees, PWN infected pine tree
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Introduction
Pine wilt disease (PWD) is currently the most devastating coniferous forest disease in the world and has been proven to be caused by an infection from the pine wood nematode (PWN),
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Bursaphelenchus xylophilus (Steiner & Buhner, 1934) Nickle (Futai, 2013). Since first reported in
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Nagasaki, Japan in 1905, this disease has spread to other East Asian countries, including China in In addition, PWN infected
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1982, Taiwan in 1985, and Korea in 1988 (Futai, 2013; Suzuki, 2002).
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pine trees were observed in the Iberian Peninsula, Portugal in 1999 (Futai, 2013; Mota et al., 1999) and then were observed in the Cáceres Region of Spain near Portugal in 2008 (Abelleira et al.,
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2011). The disbursement of the PWN within a local area is mediated by several pine sawyer However, it is known that the occurrence of PWD
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beetle species in the genus Monochamus spp.
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in remote areas or other continents are caused by an artificial transport of the PWN and its vector infected trees (Futai, 2013). Therefore, the best way to currently prevent a widespread outbreak
Concurrently, locating and burning the PWN infected trees is one of the most
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uninfected areas.
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of PWD is to prevent the transportation of pine trees from the PWN-infected areas to other
effective ways to prevent the spread of the PWD since the elimination of infected trees reduces the density of the vectors (Futai, 2013).
Various research efforts have been conducted in order to detect and confirm the PWN and its infected pine trees.
Prior to developing diagnostic methods using molecular markers,
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confirmation of the PWN infection of pine trees was based on the microscopic observation of extracted nematodes in pine trees (Futai, 2013; Yik and Birchfield, 1981). Diverse molecular markers including mitochondrial genes (Cheng et al., 2008; Kanzaki and Futai, 2002), internal
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transcribed regions (Cheng et al., 2008; Kanzaki and Futai, 2002), and various micro-satellites (Jung et al., 2010; Mallez et al., 2013) were identified and used to develop techniques.
However, most
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of the molecular tools required sophisticated machines and sometimes it was necessary to separate PCR products on agarose gels to finish a diagnosis.
To overcome these limitations,
However, the LAMP reaction mixtures must be incubated
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(Kang et al., 2015; Kikuchi et al., 2009).
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several methods using loop-mediated isothermal amplification (LAMP) have been developed
for at least one h at approximately 60°C, and strong fluorescence signals must be confirmed by
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UV illumination or other methods, in addition to requirements of extractioin genomic DNA from
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wood pellets.
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To overcome those limits, rapid testing kits using antigen-antibody interaction for quick In a previous study, we reported that Expansin B3
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diagnosis in the field must be developed.
(EXPB3) is a protein secreted from PWNs when they are infecting pine trees (Lee et al., 2012).
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this study, a monoclonal antibody (Mab) secreting fusion hybridoma library for EXPB3 was constructed and Mabs selectively recognizing PWN-infected pine trees (PWNIPTs) were screened. In addition, specificities and sensitivities of Mabs were confirmed by using various PWNIPTs collected from nine regions in Korea. 4
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Material and Methods
The target protein identification and synthesis of peptides for antigens
In previous study, we reported that ExpansinB3 (EXPB3) was highly expressed in
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propagative and dispersive stages in PWN (Lee et al., 2012). The amino acid sequence of EXPB3
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was used to blast using a B. xylophilus database in GeneDB
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(http://www.genedb.org/Homepage/Bxylophilus) and NCBI Blast
Based on the blast results, two
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(https://blast.ncbi.nlm.nih.gov/Blast.cgi) (data not shown).
PWN and other nematode species.
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peptides were designed showing amino acid sequence discrepancy to various EXP homologues in The sequences of the two synthesized peptides were 42The two peptides were
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CGLDIANLSAGVSSKLFD-59 and 141-KPATITYLKCSDTTPTTNC-150.
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conjugated with a keyhole limpet hemocyanin (KLH) and then used as antigens for immunizing
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four Balb/C mice (Peptron, Daejeon, Korea).
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Immunization and primary screening of EXPB3 specific monoclonal antibodies
The fusion hybridoma cell line constructing experiment was approved by experimental animal ethic committees at Hallym University (No. HMC2016-00622-7). We followed the standard monoclonal antibody (Mab) generation protocol that was used in Lee et al. (Lee et al., 2011). supernatants of fusion hybridoma recognizing two peptide antigens were screened by using 5
The
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enzyme linked immunosorbent assay (ELISA). Antigenic peptides (0.1 g/ml) were used to coat 96-well plates, which were then blocked with 5.0% non-fat-dried milk in PBS for one h at room temperate (RT) with shaking. After three wash cycles with 0.1% Triton X-100 in PBS (PBST), the
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supernatants of the 1920 fusion hybridoma cell lines were added and incubated at four °C overnight. After three wash cycles with PBST, the peroxidase conjugated anti-mouse IgG, A, and
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M antibodies (ThermoFisher Scientific, Waltham, MA, USA) were applied and incubated at RT for two h. One step-Ultra TMB solutions (ThermoFisher Scientific) were used to detect the
Optical densities (ODs) in each well were measured at 450 nm.
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Winooski, VT, USA).
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peroxidase activities which were then measured by using an Epoch ELISA plate reader (Biotek,
Wood pellets from 74 PWNIPT and 22 healthy pine trees (HPT) were collected by using a
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wooden pellet collector (FJ Technology Co., LTD, Seoul., Korea) from nine regions or three regions, respectively, in Korea (Supplementary Table 1). Collected wooden pellets were immediately
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transferred to the laboratory and then kept in refrigeration at four °C until used.
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To determine the optimal extraction solution, we used dH2O alone or with 0.1% Triton X100 (H2OT) and phosphate buffered saline (PBS, 0.01 M sodium phosphate, 0.154 M NaCl, pH 7.2) alone or with 0.1% Triton X-100 (PBST).
A total of five ml of four different extraction solutions
were mixed with 0.5 g of wooden pellets and incubated for five min at RT, and the solutions were then carefully decanted to avoid debris. Among the solutions, the PBS extracts displayed higher
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immunoreactivities to selected supernatants (Fig. 1).
Therefore, all other experiments were
performed using PBS as an extracting solution in this study.
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Secondary screening with the pooled PBS extracts of 74 PWN infected pine trees
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The selected 96 hybridoma lines were further screened by performing ELISA as described This screening
Thirteen clones with high ODs were selected for single cell cloning.
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Single cell cloning by limited dilution
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was done in three iterations.
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above except for using the pooled PBS extracts of 74 PWNIPT as immunogens.
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Limited dilution was performed as previously described to obtain single cell clones of 13 hybridoma lines showing a higher OD to pooled PWNIPT (Lee et al., 2011).
The supernatants The
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from the limited diluted clones were screened using pooled PBS extracts of the 74 PWNIPT.
as controls.
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PBS extracts from 22 healthy pine trees (HPT) from three regions in Korea were pooled and used The ODs of each Mab to the pooled PBS extracts of 74 PWNIPT were normalized by
the ODs of the same Mab to the pooled PBS extracts of 22 HPT.
The equation is as follows:
Normalized specificity = (an OD of the pooled PBS extracts of 74 PWNIPTs – an OD of the pooled PBS extracts of 22 HPTs)/an OD of the pooled PBS extracts of 22 HPTs.
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Measuring the normalized specificity of EXPB3-Mab-3-1-H3-C11 to 74 PWNIPTs
PBS extracts of 74 PWNIPTs from nine different regions in Korea were used since there
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may be unknown variations among PWNIPTs according to the regions and stages of infections. The
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The hybridoma cell culture medium was used for the control and background signals.
Wood pellets from 42 red pine trees and 32 black pine trees were used in this study
(Supplementary Table 1).
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screenings.
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selected hybridoma line, EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) was used for further
The degree of progression of the PWD in pine trees used in the study
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ranged from normal (zero) to completely dead (six) by visual inspections and presence of the
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PWN. Additionally, the perimeter of infected trees was measured at 1.2 m above ground and
Statistical analysis
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was used for correlation analyses (Supplementary Table 1).
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The differences between the groups were analyzed by using Analysis of Variance (ANOVA) with the Excel program (Microsoft, Redmond, WA, USA).
Tukey-Kramer post hoc analysis was
performed in order to determine differences among groups by using the Excel program (Microsoft). The Pearson correlation coefficient among ODs, degree of disease progress, and the perimeter of trees at 1.2 m above ground were calculated by using the Excel program (Microsoft). 8
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If Pearson correlation coefficient is 0.1 to 0.2 or -0.1 to -0.2, the correlation is considered low.
If
the Pearson correlation coefficient is 0.2 to 0.4 or -0.2 to -0.4, the correlation is considered medium.
If Pearson correlation coefficient is 0.4 to 1.0 or -0.4 to -1.0, the correlation is
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considered high.
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Results
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Primary screening using antigenic peptides identified 96 hybridoma lines secreting EXPB3
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antibodies
The EXPB3 monoclonal antibody (Mab) library containing 1,920 fusion hybridoma lines
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was generated for screening Mabs that recognized PWNIPTs. The primary screening was performed to identify fusion hybridoma lines secreting EXPB3 peptides recognizing Mabs which We normalized the ODs of each 96-well plate by using the
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were used as antigens by ELISA.
highest OD in each plate because there were some variations among the plates. The cut-off
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value of normalized OD obtained from ELISA was > 40% of the highest OD for each plate. After primary screenings, 96 fusion cells were selected for further study (Supplementary Fig. 1).
PBS was a suitable solution for extracting antigens for EXPB3-Mabs
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Selecting solutions that can most efficiently extract the antigens is one of the important steps for the development of a diagnostic technology.
Particularly, it is important to select an
extraction solution that does not contain health or environmental hazard chemicals because the
harmful to humans or the environment.
Therefore, we choose dH2O and PBS with or without 0.1%
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The LD50 of Triton X-100 is 1,800 mg/kg when administered orally to rats (MSDS,
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Triton X-100.
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diagnosis method used in the field could be problematic if the solutions contain substances
exposure to humans and other wild animals.
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2013). Thus, an addition of 0.1% Triton X-100 to solutions would not cause a severe toxic
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Four EXPB3-Mab3-1-H3-C11 sub-clones were used for performing ELISA with 4 different extracts to compare the extraction efficiency of the four solvents (Fig. 1). Among the four EXPB3-
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Mabs, EXPB3-Mab-3-1-H3-C11-E3 and –E4 had higher ODs when compared with two other Mabs. Although the ODs were different among the four Mabs, they displayed similar OD trends to the PBS extracts from PWNIPTs from Pyeoson, Jeju-do
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four extraction solutions and three PWNIPT.
or PBST.
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and Iksan, Jellabuk-do displayed relatively high ODs when compared with extracts with H2O, H2OT, However, dH2O extracts from a PWNIPT from Uiryeong, Gyeongsangnam-do displayed
higher ODs than other extracts (Fig. 1).
These results suggest that PBS may be suitable for
extracting antigens from PWNIPTs.
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Nine hybridoma lines showed specificity to PBS extracts of PWN-infected pine trees
The PBS extracts of PWNIPTs were used to analyze specificity of each hybridoma by ELISA in order to identify hybridoma lines secreting Mabs capable of recognizing PWNIPTs among the We obtained 74 PWNIPTs containing 42 red pine trees and 32 black
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96 selected hybridoma lines.
The OD of the 96 hybridoma lines
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74 PWNITs were pooled and used for secondary screening.
The PBS extracts from the
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pine trees from nine PWN affected regions (Supplementary Table 1).
were normalized by using EXPB3-Mab-3-3-D9, which has the highest OD. Nine hybridoma lines
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revealed a normalized OD value above 0.2 (Fig. 2).
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Single clones from EXPB3-Mab-3-3-D9 and -3-3-H3 had high normalized specificity to
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PWNIPTs
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To established single cell clones, cells in 13 lines including nine lines with OD value > 0.2 had limited dilution on 96 well plates.
The supernatants from the 96 wells were screened by
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using pooled PBS extracts of 74 PWNIPTs and 22 HPTs to calculate normalized specificities. Among 13 lines examined, two lines, EXPB3-Mab-3-1-C9 and -3-2-G11, failed to divide and could not execute ELISA. Single cell clones from five hybridoma lines including EXPB3-Mab-3-2-E1, EXPB3-Mab3-2-G9, EXPB3-Mab-3-2-F1, EXPB3-Mab-3-2-E1, and EXPB3-Mab-3-1-G4 displayed very low normalized specificities (< 1.0) (Supplementary Fig. 2E–I). 11
Single cell clones from EXPB3-
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Mab-3-1-C3, EXPB3-Mab-3-1-F4, EXPB3-Mab-3-1-A5, and EXPB3-Mab-3-2-E9 displayed medium normalized specificities ranging between 1.04~2.46 (Supplementary Fig. 2A-D).
Single clones from two lines, EXPB3-Mab-3-3-D9 (EXPB3-D9) and EXPB3-Mab-3-1-H3
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(EXPB1-H3) had a higher normalized specificity than those from other lines (Fig. 3).
Three of the Those
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single cell clones of EXPB3-D9 and EXPB1-H3 had a normalized specificity of 3.4 or more.
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clones were used for further studies.
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Immunoreactivity variations in PWNIPT revealed by EXPB3 antibodies
The extent of the disease progression in PWNIPTs used in this study was determined only Therefore, there is no
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by the external characteristics of the trees at the time of pellet harvesting.
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information regarding the detailed infection period or physiological change for each pine tree.
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We used the developed EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) to measure the amount of antigens present in each of the 74 PWNIPTs collected from the nine different locations (Fig. 4A).
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We compared the ODs of EXPB3-C11-E4 for the red pine trees with those of the black pine trees and found that the ODs of the black pine trees were statistically significantly higher than those of the red pine trees (Fig. 4B).
Black pine trees collected from three regions in Jejudo
tended to have higher ODs than those of the mainland region when comparing 32 black pine trees collected from five regions in Korea (Fig. 4C). 12
However, there was no statistically significant
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difference among the 42 red pine trees collected from seven regions in Korea (Fig. 4D).
There was low correlation between the ODs and degree of disease progression for the red pine trees (Pearson correlation coefficient = -0.174). However, there was a medium degree of
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correlation between the perimeter of the trees at 1.2 meters above ground and the degree of
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disease progression as well as between the perimeter of the trees at 1.2 meters above the ground In contrast,
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and the ODs (Pearson correlation coefficients: -0.21529 and 0.34562, respectively).
there was high correlation between ODs and the degree of disease progression as well as
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between ODs and the perimeter of the trees at 1.2 m above ground (Pearson correlation However, there was medium
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coefficients: 0.49583 and 0.43023, respectively) for black pine trees.
correlation between the degree of disease progression and the perimeter of the trees at 1.2 m The results of this
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above ground (Pearson correlation coefficient: -0.21069) for black pine trees.
analysis suggested that there is a difference in EXBP3-Mab reactivity depending on the species,
Discussion
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distribution area and developmental stage for individual black pine trees.
The major goals of this study were to generate an array of EXPB3-Mabs that can be used for the rapid diagnosis of PWNIPTs in the field and establish a method for collecting wooden pellets in order to complete an accurate diagnosis. 13
To accomplish this goal, we first identified
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the suitable solutions that can extract the antigens most efficiently from the pellet of the PWNIPTs. We used the PBS extracts of the PWNIPTs to identify the most specific Mabs.
EXPB3-Mabs were
screened using not only for the high reactivity to the PBS extracts of PWNIPTs but also for the low
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reactivity to the PBS extracts of HPTs to increase the accuracy and sensitivity of EXPB3-Mabs. Therefore, the EXPB3-Mabs were selected based on a normalized sensitivity calculated with the
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Methods section methodology might have high specificity and sensitivity to the PWNIPTs.
Since other research papers report on methods for detecting a PWN from the PWNIPTs,
We considered the physical characteristics of the Korean adults, and the
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Schröder et al., 2009).
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pellets of PWNIPTs collected from trunks at 1.3 m above ground (Magnusson et al., 2007;
sampling position was determined to be located at 1.2 m above ground.
This methodology was
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applied to all the samples similarly to increase the possibility of application in the field.
Our research concluded that the black pine trees among the examined PWNIPTs showed
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statistically significant higher reactivity to EXPB3-Mabs than the red pine trees (Fig. 4B).
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Additionally, the reactivity for the black pine trees displayed a significant correlation with the degree of disease progression and the perimeters of the trees.
This result suggests that, in
contrast to red pine trees, the PWD progression in black pine trees can be predicted by the reactivity to the EXPB3-C11-E4 antibody.
EXP is an enzyme present in plants and has a significant role in mediating cell wall
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loosening, and it reduces the adhesion between adjacent cell wall polysaccharides to enable normal development (Marowa et al., 2016).
In addition, the EXPs of the plant parasitic
nematodes have a function similar to the EXPs of the plants, and therefore, they might assist the
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invasion of nematodes into the host plants (Qin et al., 2004). The EXP3-Mabs developed in this study will be an invaluable resource not only for a developing rapid detection tool, but for also
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studying the mechanisms underlying the invasion and propagation of PWN in pine trees.
Although the various genetic makers that can identify PWNs have been reported (Cheng
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et al., 2008; Futai, 2013; Jung et al., 2010; Kanzaki and Futai, 2002; Mallez et al., 2013), protein
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markers for detecting PWNIPTs were not reported before our investigation. Recently, the secreted proteins from B. xylophilus when they were cultured in pine wood extracts were reported
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(Shinya et al., 2013). However, no study has been done to determine whether these secreted proteins are actually present in PWNIPTs. Since our data provided strong evidence that EXPB3
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was present in PWNIPTs, EXPB3 is the first protein biomarker for detecting PWNPITs.
Further
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researches in developing rapid diagnosis kit will allow us to do on-site diagnosis which can be completed within several min.
Acknowledgement 15
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This work was supported by a research grants from National Institute of Forest Science (Title: Development of Bursaphelenchus xylophilus detection techniques by using its specific antigens No. 12178-0279-01).
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and antibodies.
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Figure legends
Fig. 1. EXPB3-Mabs displayed a relatively high immunoreactivity to PBS extracts. Compared with H2O, H2O with 1.0% Triton X-100 (H2OT), or PBS with 1.0% Triton X-100 (PBST) extracts, PBS The PBS extracts from two PWNIPTs obtained from
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extracts revealed high optical densities (ODs).
However, the H 2O
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Pyeoson, Jeju-do and Iksan, Jellabuk-do had higher ODs than other extracts.
The immunoreactivity of 96 selected hybridoma to PBS extracts from PWNIPTs.
PBS
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Fig. 2.
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extracts from a PWNIPT from Uiryeong, Gyeongsangnam-do had higher ODs.
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extracts from 74 PWNIPTs collected from 9 regions of Korea were pooled and used.
Fig. 3. Limited dilution results of two EXPB3 hybridoma clones.
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To select single clones from 13 EXPB3 hybridoma lines, limited dilutions were performed. (A) The
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normalized specificities of 60 supernatants from the limited diluted cells from EXPB3-Mab-3-1-D9. (B) The normalized specificities of 48 supernatants from the limited diluted cells from EXPB3-Mab3-1-H3.
Fig. 4.
Three clones from each line were selected for further analysis.
Immunoreactivity of the EXPB3-C11-E4 to PWNIPTs. 17
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(A) The map of Korea showing nine locations where 74 PWNIPTs were collected. (B) Comparison of
immunoreactivity
between
PWN-infected
red
and
black
pines.
(C)
Comparison
of
immunoreactivity to PWN-infected black pines among different collection sites. (D) Comparison of
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immunoreactivity to PWN-infected red pines among different collection sites.
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Fig. 2. 20
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Fig. 4.
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Reference Abelleira, A., Picoaga, A., Mansilla, J.P., Aguin, O., 2011. Detection of Bursaphelenchus xylophilus, causal agent of pine wilt disease on Pinus pinaster in Northwestern Spain. Plant Dis. 95, 776. Cheng, X.-Y., Cheng, F.-X., Xu, R.-M., Xie, B.-Y., 2008. Genetic variation in the invasive process of
Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae) and its possible spread routes in China. Heredity 100, 356-365. Futai, K., 2013. Pine wood nematode, Bursaphelenchus xylophilus. Annu. Rev. Phytopathol. 51, 61-
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Graphical abstract
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Highlights 1.
EXPB3 specific Mabs were screened from 1920 clones
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Among them, 13 clones were limited diluted to select single clones
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EXPB3-1-H3 clones showed highest specificity to pine wood nematode infected pine trees (PWNIPT) There were differences between red pine trees and black pine trees in EXPB3 Mab
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immuno-reactivity.
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These EXPB3 Mab could be used to detect PWNIPTs at the field
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5.
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