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ISSN : 1226-9999(Print)
ISSN : 2287-7851(Online)
Korean J. Environ. Biol. Vol.42 No.4 pp.582-592
DOI : https://doi.org/10.11626/KJEB.2024.42.4.582

A survey on occurrence and distribution of plant-parasitic nematodes in citrus orchards

Karthi Natesan, Sekeun Park, Hyoung-Rai Ko, Eunhwa Kim, Sohee Park, Byeong-Yong Park*
Crop Protection Division, National Institute of Agricultural Sciences, RDA, Wanju 55365, Republic of Korea
*Corresponding author Byeong-Yong Park Tel. 063-238-3316 E-mail.
daggernema@korea.kr

Contribution to Environmental Biology


▪ The present investigation contributes to environmental biology by revealing the prevalence and ecological impact of plant -parasitic nematodes (PPNs) in citrus orchards.


▪ Our finding underscores the need for proper control measures to protect both agricultural and natural ecosystems.


15/11/2024 11/12/2024 19/12/2024

Abstract


Current investigations provide a comprehensive understanding of the occurrence and biodiversity of plant-parasitic nematodes (PPNs) in the major citrusproducing regions of Jeju Island, South Korea. Our survey identified five genera and five species of PPNs from 82 infested Citrus unshiu field samples collected across 116 sites in the Jeju provinces. Community analysis revealed the highest prevalence of PPNs (39.02%) at Namwon-eup, significantly driven by Tylenchulus semipenetrans, followed by Paratylenchus sp., Helicotylenchus sp., Meloidogyne sp., and Pratylenchus sp. Data indicate that all 82 sites were infested with T. semipenetrans (70.68%), marking a considerable increase in prevalence compared to previous surveys and posing a significant threat to citrus cultivation. The study results also demonstrate the influence of soil type on PPNs communities, revealing correlations between soil texture and nematode diversity. Citrus orchards cultivated in black clay loam soil exhibited significant PPN infestations. Overall, the PPN survey underscores the economic importance of monitoring citrus nematode infection rates and maintaining economic threshold levels in citrus production. It also emphasizes the need for developing effective management strategies to control PPNs, which are essential for maintaining crop yield and ensuring agricultural sustainability.


prevalence , plant parasitic nematodes (PPNs), , Tylenchulus semipenetrans

초록

    1. INTRODUCTION

    Worldwide, citrus fruits have significant socioeconomic importance in horticulture. Citrus unshiuMarc. cv. Miyagawa-wase has been a common cultivar in Jeju Island and is highly valued among the world’s top sweet citrus varieties, with a considerable contribution to global citrus production (Kim et al. 2020). A survey conducted in 2023 reported that a maximum manda- rin production is around 15.8 kha in Jeju Island (Heo et al. 2023a). Usually, Satsuma mandarin can sustain at low temperatures, making its widespread cultivation in Korea and also in subtropical climates regions like Jeju Island (Moon et al. 2015;Kwon et al. 2021;Kim et al. 2023).

    For decades, plant parasitic nematodes (PPNs) have been considered as a serious threat to citrus fruit production. The USDA Foreign Agricultural Services reported that both biotic and abiotic stressors substantially reduced annual yields of Jeju mandarins from 2022 to 2023 (USDA 2022, 2023). Similar studies reported about a 10% citrus yield loss due to dieback diseases caused by PPNs infestation. Although it is widely accepted that the decrease in yield was associated with PPNs, the nematode-related issue has been proven to be a challenging task due to the co-existence of different species of nematodes in the same field and complications in diagnostic traits. PPNs have been reported and documented having significantly influence on the nation’s economy through substantial yield losses. Some literature has promisingly associated plant parasitic nematodes such as root-knot nematodes (Meloidogyne incognita), burrowing nematodes (Radopholus similis), and citrus nematodes (Tylenchulus semipenetrans) with common citrus field infestations, accounting for about a 30-40% reduction in yield. Furthermore, PPNs infection aggravates disease complexes and impairs resistance mechanisms in specific fruit crop varieties (Ford 1954;Keshari and Mallikarjun 2022).

    T. semipenetrans, can potentially infest citrus orchards growing on fine-textured or sandy soils rich in organic matter, especially in regions with fluctuating soil salinity (Timmer et al. 2003). Studies showed that T. semipenetrans can tolerate extreme climatic conditions in tropical and subtropical regions. Notably, the sustainability of T. semipenetrans in Florida and California (USA, 24-60%), Brazil, and Spain (70-90%) under a wide range of extreme environmental conditions reveals its adaptability to diverse environments (Park et al. 2009;Abd-Elgawad et al. 2015). South Korea is one such land with extreme climatic conditions, and our earlier report on the occurrence and distribution of PPNs encountered T. semipenetrans infestation in citrus fields along with a few other PPNs such as Pratylenchus sp., Helicotylenchus sp., Meloidogyne sp., Xiphinema sp., and Scutellonema sp. (Park et al. 2009). However, there is a significant lack of recent data on PPNs distribution in Jeju Island, highlighting the knowledge gap.

    Thus, a current survey study was conducted and aimed to investigate the incidence of PPNs associated with citrus orchards, along with an integrative molecular taxonomic approach for species identification and categorization of PPNs. Additionally, the study included an assessment of nematode infestation severity assessment by providing detailed survey data on the current prevalence and distribution of citrus nematodes in Jeju Island. This research effectively underscores the necessity for further studies to develop effective management strategies against these soil-borne pathogens.

    2. MATERIALS AND METHODS

    2.1. Survey site and soil sampling

    The survey was conducted in the citrus plantations of Jeju-do province, South Korea, between June 2022 and June 2023 (Fig. 1, Supplementary Table S1). The study involved random sampling of 116 sites, with approximately 10 samples collected at each site (crisscross pattern method). The soils were mixed thoroughly before being stored in polyethylene bags and were transported to the Nematology Laboratory, NAAS, RDA. All samples were stored in a cold room (8°C until nematode extraction and processing (Fleming et al. 2016).

    2.2. Nematode extraction

    Collected soil samples were blended in a 70 cm×20 cm plastic bag, and 100 cm3 of soil was added to a plastic container containing 4 L of tap water. The mixture was thoroughly mixed and left to settle for 15 seconds, followed by a modified Cobb’s decanting and sieving (#20 and #400 mesh) method for nematode extraction (Van Bezooijen 2006). Later, the nematode population in the sieved soil suspension was allowed to settle for 48 h in a modified-Baermann funnel and finally collected in 15 mL tubes (Baermann 1917).

    2.3. Morphometric and molecular identification of PPNs

    Based on morphological features described earlier, extracted PPNs were identified, enumerated, and recorded at the genus level in Syracuse counting dishes under a dissecting stereomicroscope (nematodes per 100 cm3 soil) (Park et al. 2009). Later, DNA from a single nematode sample was used for the species-level identification. Molecular amplification of the D2- D3 segments of 28S rRNA was performed using D2A and D3B-primer sets (De Ley et al. 1999;Subbotin et al. 2001, 2006). Sequences of the PCR amplicons were subjected to Bayesian inference (BI) phylogenetic anal ysis using MrBayes 3.2.7 (Sandoval-Ruiz et al. 2023).

    2.4. Assessment of PPNs community structure

    The PPNs community structure was calculated (Table 1, Supplementary Table S2). Additionally, the study aimed to investigate the influence of soil type on PPNs diversity, based on earlier report by Seok Chan Koh which classified the collected soil sample based on texture (Oh et al. 2019). All processing methods for the raw data and the statistical calculations were performed as mentioned (Supplementary Table S2). A heat map analysis with a dendrogram representing the cluster of PPNs genus was performed using XLSTAT 2016 (Addinsoft, Inc., France). The graphical representations of PPNs community structure were made using Graph- Pad Prism version 5.0 and Origin (Pro) version 9.0, USA.

    3. RESULTS

    3.1. Occurrence and distribution of PPNs

    The occurrence and geographical distribution of PPNs infestation in citrus orchard fields of Jeju Island were surveyed between 2022 and 2023. The analyzed results identified 5 PPNs genera and 5 PPNs species. The survey defined 82 positively PPNs-infested sites out of 116 citrus orchard field samples collected (Fig. 1, Supplementary Table S1). For the study’s purpose, the collected data were segregated into 29 localities across 6 districts of Jeju Island. The maximum PPNs distribution was recorded in Namwon-eup (39.02%), followed by other regions as shown (Fig. 1b). Based on morphological traits, the occurrence of Tylenchulus sp. in all the collected samples was identified. Along with the Tylenchulus sp., other PPNs such as Helicotylenchus sp., Paratylenchus sp., Meloidogyne sp., and Pratylenchus sp. infections were also recorded (Fig. 1d). The maximum density of Tylenchulus sp. was 485 nematodes/100 cc of soil, followed by Meloidogyne sp. (190), Helicotylenchus sp. (100), Paratylenchus sp. (15), and Pratylenchus sp. (10), respectively (Supplementary Table S3). The statistical analysis showed a 13.18% as a high relative density for Tylenchulus sp. at Namwon-eup with a mean density (MD) of 99.84, followed by Pyoseon-myeon (MD: 82.67%), respectively (Table 1).

    3.2. Community structure and the prevalence of PPNs in citrus orchards

    The community structure and distribution of PPNs across six regions of Jeju Island showed that Tylenchulus sp. had the highest relative density (RD) along with a significantly high prevalence ranging from 55.56% to 100%, whereas other PPNs generas were comparatively low prevalence as described (Table 1, Supplementary Table S4). Portrayed results (Table 1) reflected a comparatively high relative frequency (RF) and greater relative prominence value (RPV) for Tylenchulus sp., in Namwon-eup (RPV=32.98%, PV= 901.11). Overall community structure analysis among all six districts of Jeju Island showed high prominence for Tylenchulus sp. as shown (Table 1, Supplementary Table S4).

    Moreover, RF for all the 82 sample sites showed high percentile values, which coincides with the hierarchical clustering heat map depicting PPNs incidence and population density (PD) (Fig. 2a). The dendrogram reflected 5 PPNs genera clustered according to distribution patterns among the collected 82 samples from the citrus orchards. Furthermore, difference in Heatmap intensity reflected PD at all 29 locations, showing Tylenchulus sp. as the dominant PPNs, particularly in Namwon-eup and Pyoseon-myeon. Cluster analysis formed a distinct single clade for Tylenchulus sp., while other four taxa such like Helicotylenchus sp., Paratylenchus sp., Meloidogyne sp., and Pratylenchus sp. were grouped to form another clade reflecting a high similarity and showed great distance from each other as illustrated (Fig. 2b). And, Tylenchulus sp. showed the highest abundance of 47.09% in Namwon-eup. Additionally, the obtained re- sult demonstrates the strong correlation between abundance and RF of Tylenchulus sp. particularly in Namwon- eup and Pyoseon-myeon regions (Fig. 3, Supplementary Table S4). Although Helicotylenchus sp. were found less PD when compared with Tylenchulus sp., but this genus showed a considerable abundance in regions like Pyoseon-myeon, Namwon-eup, and Seongsan-eup, and these results were highly coincided with cluster analysis (Supplementary Table S4).

    The occurrence and distribution of PPNs in citrus fields are highly influenced by soil variables, including soil texture. This survey categorized six soil textures: BLCLS, BLSLS, DBCLS, DBSLS, BCLS, and BSLS (Table 2, Supplementary Table S6). The represented box plot of relation between PPNs in citrus field and soil shows higher mean abundance of PPNs in black and dark brown clay loam soils followed by sandy loam soils as shown (Fig. 4). These results obtained were highly coincided with the frequency and density of PPNs analyzed. Subsequently, few infections of Melodogyne (Mel)in BLCLC and Pratylenchus (Pra) in BLSLS were also recorded, indicating variability in nematode communities based on soil conditions and emphasizing the need for further investigation into the factors influencing these dynamics for effective PPNs management.

    3.3. Molecular characterization of PPNs

    The sequence results of 90 PPNs isolated from citrus were subjected to molecular characterization using D2A/D3B primer sets and were depicted in a phylogenetic tree (Fig. 5). All 90 PPNs sequences were deposited in GenBank (Supplementary Table S6). The Bayesian consensus tree revealed five distinct clades where all the 82 T. semipenetrans formed a single clade including reference sequences acquired from NCBI. Paratylenchus sp. formed a second clade and identified as Paratylenchus lepidus. Furthermore, accession PP789631 and PP789630 were identified as of Helicotylenchus dihystera, and the sub-branch PP789653 was identified as Pratylenchus loosi. The analysis used Psilanchus sp. and Coslenchus costatus as outgroups to root the tree.

    4. DISCUSSION

    4.1. Occurrence and distribution of PPNs in citrus field

    The ecosystem drives on the key principle of biodiversity, incorporating vital elements such as ecology, biogeography, evolutionary and physical processes that potentially interact with one another (Huston 1997). Recent studies have highlighted the significance of nematodes in Korean agricultural environments, emphasizing their role in understanding microbial dynamics within plant ecosystems (Ko et al. 2023). And their findings provided insights into the nematode community structure and its potential implications for agricultural and ecological investigations in Korea (Heo et al. 2023b). In context, the present survey report provides a comprehensive understanding of PPNs diversity in citrus orchards on Jeju Island. Through morphological trait and molecular analysis, four genera and five species were identified from 82 infested samples from Jeju Island. The molecular characterization using Bayesian tree confirmed that 5 distinct species including T. semipenetrans, Paratylenchus lepidus, Helicotylenchus dihystera, Pratylenchus loosi and Meloidogyne sp. were infesting the surveyed citrus field. Since low densities of Meloidogyne nematodes were detected in this study, further sampling and molecular identification will be necessary to fully understand the distribution and prevalence of this species. Most of PPNs infestation in the surveyed citrus fields was T. semipenetrans, as stated in our previous study from 2009 (Hayasaka et al. 1988;Park et al. 2009;Rashidifard et al. 2015). Numerous survey across Asian countries (Lin et al. 2016;Wu et al. 2023;Yamaga and Unno 2023), and other parts of the world reported its occurrence in the citrus fields (Abd-Elgawad 2020). And the current survey documented a significantly notable prevalence of T. semipenetrans, with a high mean density (MD) of 485 nematodes/ 100cc of soil, showing a significant increase compared to our earlier reported survey (MD: 328.3/100cc of soil) (Park et al. 2009). In addition to Korean citrus fields, a similar infection rate of T. semipenetrans has been well documented in major citrus-growing regions, including the USA (24-60%), Brazil, and Spain (70- 90%), Turkey (21.9%), Morocco (57-88%), China (up to 90%), and Japan (90%), among others (Van Gundy and Meagher 1977;Heald and O’Bannon 1987;Duncan 2005;Duncan et al. 2019;Uludamar 2022;Zoubi et al. 2022;Yamaga and Unno 2023).

    4.2. Economic threshold of T. semipenetrans

    T. semipenetrans infestation significantly affects citrus yield, especially under various edaphic and environmental factors. Many case studies have reported T. semipenetrans as a primary cause of slow decline diseases, leading to severe deterioration of citrus orchards. According to earlier reports, the economic threshold (ET) level was formulated using the estimated cost of damage at a certain level of nematode population, which is the same as the value required to control the nematode infestation (Ferris 1978). As mentioned, the ET level for the T. semipenetrans infestation was 330- 350 in Mandarin fields of Spain between 2003 and 2004 (Sorribas et al. 2008). Additionally, an Israeli citrus field was reported over 4,000 females/gm of roots for T. semipenetrans, with an estimated ET was around 1,000/100cc of soil (Bridge and Starr 2007). Based on the ET level, the values lower than 300 are indicated as low, values exceeding 700 as moderate, and those above 1,400 represent a high threat to the socioeconomic status (Abd-Elgawad 2020). In our study, the mean density of T. semipenetrans in the citrus field was 485/100cc of soil, indicating a near-moderate ET level. Furthermore, the present study spotlighted the high density of T. semipenetrans in both Jeju Island, and this data would reflect with high ET for the citrus production in South Korea.

    4.3. Community structure and the prevalence of PPNs in citrus orchards

    Briefly, understanding T. semipenetrans prevalence in the citrus orchards holds the major economic importance for maintaining ET level of citrus production. Generally, the Tylenchulus sp. are more prevalent and abundant taxa and have a greater potential to migrate to nearby citrus fields. Consequently, widespread distribution would result in increased abundance (Pandey 2023). The reasons behind the widespread occurrence of T. semipenetrans have been well studied including, infected seedlings, contaminated plant composite and organic matter, agricultural machinery, and irrigation methods (Abd-Elgawad et al. 2016a). This analysis revealed that Tylenchulus sp. had a prevalence of over 55% in all surveyed regions, as mentioned in the results (Table 1). The continuous increase in Tylenchulus sp. prevalence compared to our previous data might be influenced by the notable fluctuations in the subtropical climatic conditions of Korea and the resistance of citrus nematodes to the previously available management methods (Park et al. 2009). Next to Tylenchulus sp., the distribution of Paratylenchus sp. was notably high in the Namwon-eup. Similar co-occurrence of along with Tylenchulus sp. in citrus fields was documented in other countries as well (Abivardi 1970;Abd-Elgawad et al. 2016b;Zoubi et al. 2022). Apart from those two nematodes genera, Helicotylenchus sp. was also found in Namwon-eup and Aewol-eup of Jeju Island, though with lesser relative prevalence (PR). Also, numerous literature reports from the past few decades had stated the occurrence of Helicotylenchus sp. in the citrus cultivation areas around the world (Abd-Elgawad 2020). Interestingly, the current study detected a Meloidogyne sp. in the Citrus unshiu growing field at Namwon-eup of Jeju Island, with a PV of 6.87 and RF 100%, respectively. This study highlights the occurrence of Meloidogyne sp. which was not reported in our previous survey, and similar occurrences were rarely reported in a few other countries (Duncan 2009). Root lesion nematodes of Pratylenchus sp., were also detected in negligible density at one location, and measures should be taken to control their migration to other citrus orchards. Similarly, Brazil had accounted for 1% of total citrus nematode infestation, less than 1% in Morocco citrus, whereas the majority of citrus fields in Florida detected with root lesion nematodes (O’Bannon and Tomerlin 1973;De Campos et al. 2002;Bucki et al. 2020). Following this, cluster analysis results based on PPNs communities in the citrus soils and the obtained dendrogram significantly showed the diversity level among the different localities of Jeju Island, and the findings were highly coincided with the calculated computed community structures, indicating predominant Tylenchulus sp. infestation.

    The collaborative report from Jeju National University documented the different soil types and their influence on crop cultivations (Oh et al. 2019). In connection, with the current study, we categorized the soil based on the physical factors such as soil color and itstexture. Furthermore, the study had evaluated the influence of soil texture and the abundance of PPNs. The analysis found that black clay loam soil in Jeju Island possesses a high diversity of PPNs, followed by black sandy loam soil, particularly T. semipenetrans infestation. As described earlier, clay loam soil has been reported to have a higher abundance of citrus nematodes than fine sandy soil; hence, it directly affects the migration and distribution of nematode communities (Salahi Ardakani et al. 2014). In conclusion, this study highlights the crucial role of biodiversity in soil ecosystems and provides a better understanding of the current prevalence and diversity of PPNs in Jeju Island citrus orchards. Particularly, T. semipenetrans infestation poses significant threats to citrus cultivation. Additionally, it is important to mention the limitations of the present PPNs survey, which includes its focus on specific regions and the limited data on nematode diversity. Moreover, there is a need for further research to develop more precise strategies for nematode management, with suggested effective approaches in citrus fields.

    5. CONCLUSION

    The present study provides key findings on the current status of plant parasitic nematodes in citrus fields, highlighting the economic significance of monitoring PPNs infestation rates and managing the ET levels in citrus production. Overall, understanding the prevalence and distribution of parasitic nematodes in citrus orchards is a key factor in implementing effective management to control yield losses and sustain citrus production in South Korea and beyond.

    ACKNOWLEDGEMENTS

    This work was supported by the Rural Development Administration (RDA; Project Grant Number RS-2023- 00218522).

    CRediT authorship contribution statement

    K Natesan: Experimentation, Methodology, Writing- Original draft. S Park: Conceptualization, Methodology, Validation, Writing-Original draft. HR Ko: Writing-Review & editing, Methodology. E Kim: Formal analysis, Investigation. S Park: DNA isolation and sequence analysis. By Park: Conceptualization, Funding acquisition, Supervision, Methodology, Writing- Review & editing.

    Declaration of Competing Interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    SUPPLEMENTARY MATERIALS

    Supplementary material associated with this article can be found, in the online version, at https://doi.org/ 10.11626/KJEB.2024.42.4.582.

    Figure

    KJEB-42-4-582_F1.gif

    Geographical location surveyed for the occurrence and diversity of plant -parasitic nematodes (PPNs) in South Korea. (a) Pie chart representing the distribution of total sampling sites (TSPL); (b) Pie chart representing the PPN-infested sample sites (ISPL), as shown in Supplementary Table S1; (c) Geographical map showing the sampled sites on Jeju Island; (d) Maximum and minimum range of PPNs in the surveyed soil samples.

    KJEB-42-4-582_F2.gif

    Community structure showing the relative abundance of plant-parasitic nematodes (PPNs) in citrus fields. (a) A schematic dot plot representing the relative abundance of different genera of PPNs in the citrus-growing regions of South Korea; (b) A heat map illustrating the hierarchical clustering of PPN genera in citrus fields. The color key scale indicates normalized nematode density and prevalence (nematode density per 100 cc of soil sample). The respective abbreviations for the sampling locations on Jeju Island are provided in Table 2 and Supplementary Table S1.

    KJEB-42-4-582_F3.gif

    Two-dimensional abundance scatter plot representing the relationship between relative frequency (RF) and the abundance of plant-parasitic nematodes (PPNs). The figure illustrates the relationship between RF and the abundance of PPNs in the surveyed locations. Density is defined as the average number of nematodes per 100 cc of soil. The size of the objects represents the abundance of PPNs in a particular region.

    KJEB-42-4-582_F4.gif

    Box plot showing the abundance of plant-parasitic nematodes (PPNs) according to soil types. Data are presented as mean±SD. The box plot illustrates the distribution of the data, with the dark line inside the box representing the median, the ‘x’ marking the mean value, and the bars indicating the upper and lower quartiles. Outliers are represented as individual dots. Abbreviations: BSLS, Brown sandy loam soil; BCLS, Brown clay loam soil; DBSLS, Dark brown sandy loam soil; DBCLS, Dark brown clay loam soil; BLSLS, Black sandy loam soil; BLCLS, Black clay loam soil.

    KJEB-42-4-582_F5.gif

    Molecular characterization of PPN isolates. A Bayesian 50% majority-rule consensus tree was inferred from the D2-D3 expansion domains of the large subunit ribosomal RNA sequence alignment under the GTR+I+G method. The respective NCBI GenBank accession numbers and host details are provided in Supplementary Table S6 (Hayasaka et al. 1988).

    Table

    Community structure of plant -parasitic nematodes (PPNs) surveyed in citrus orchards

    *Tyl, Tylenchulus sp.; Hel, Helicotylenchus sp.; Para, Paratylenchus sp.; Mel, Meloidogyne sp.; Pra, Pratylenchus sp.
    Abbreviations: PD, Population density of PPNs; TSPL, Total samples per location; ISPL, Infested samples per location; PR, % prevalence; MD, Mean density of PPNs in ISPL; RD, % Relative density; AF, Absolute frequency; RF, % Relative frequency; PV, Prominence value; RPV, Relative prominence value. PPNs community structure data represented in the above table were computed based the formula (Supplementary Table S2).

    List of citrus soils surveyed for the assessment of plant-parasitic nematodes (PPNs) in citrus orchards of Jeju Island

    *Soil type: BSLS, Brown sandy loam soil; BCLS, Brown clay loam soil; DBSLS, Dark brown sandy loam soil; DBCLS, Dark brown clay loam soil; BLSLS, Black sandy loam soil; BLCLS, Black clay loam soil. Detailed sampled locations and their details were mentioned in supplementary data.

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    Vol. 40 No. 4 (2022.12)

    Journal Abbreviation 'Korean J. Environ. Biol.'
    Frequency quarterly
    Doi Prefix 10.11626/KJEB.
    Year of Launching 1983
    Publisher Korean Society of Environmental Biology
    Indexed/Tracked/Covered By

    Contact info

    Any inquiries concerning Journal (all manuscripts, reviews, and notes) should be addressed to the managing editor of the Korean Society of Environmental Biology. Yongeun Kim,
    Korea University, Seoul 02841, Korea.
    E-mail: kyezzz@korea.ac.kr /
    Tel: +82-2-3290-3496 / +82-10-9516-1611