Nematode-citrus plant interactions: host preference, damage rate and molecular characterization of Citrus root nematode Tylenchulus semipenetrans

doi:10.21203/rs.3.rs-1502604/v1

Download PDF

Research Article

Nematode-citrus plant interactions: host preference, damage rate and molecular characterization of Citrus root nematode Tylenchulus semipenetrans

https://doi.org/10.21203/rs.3.rs-1502604/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Aims

Citrus plants are hosts of numerous plant-parasitic nematodes (PPNs), which are microscopic organisms and hidden enemies for plants. Among the PPNs, the citrus root nematode, Tylenchulus semipenetrans, causes significant damage to citrus plantations in the world. Understanding the citrus nematode population, precise nematode identification, host preference among the citrus species, and damage threshold level are crucially important to control T. semipenetrans. Although some studies have been conducted, minutiae about citrus plant-nematode interactions, nematodes density, and molecular nematode identification have not been well understood.

Methods

This study aimed to reveal nematode identification using molecular and morphological methods, host-nematode interactions, host (citrus species) preference, damage of economic threshold (ET), and economic-injury level (EIL). Therefore, citrus plantations of different provinces located in the Mediterranean region of Turkey were investigated.

Results

ITS sequences revealed that samples were infested with the citrus root nematode Tylenchulus semipenetrans. The lowest nematode density was obtained from Mersin (80-second-stage juveniles(J2s)/150g-soil), while the highest density was obtained from Adana (18260-J2s/150g-soil). Out of the 73 samples collected from the Mediterranean region, 21.9% have passed the EIL, while 8.2% are on the ET. Citrus nematode reproduced utmost level on roots of Citrus reticulata, followed by C.sinensis, C.limon, and C.parasidisi, respectively.

Conclusion

This study revealed that the Citrus nematode is more common than it was thought and that the population fluctuations change according to the citrus plant species. It is of great importance to investigate the possible genetic changes in nematode populations caused by global warming and its negative impact on citrus yield.

Citrus nematode

Host preference

ITS sequence

Molecular characterization

Tylenchulus semipenetrans

Orange (Citrus sinensis), mandarin (Citrus reticulata), grapefruit (Citrus paradisi) and lemon (Citrus limon) are economically important citrus plant species. Many pests and diseases, including plant-parasitic nematodes (PPN), cause significant crop losses in citrus. Among the PPN, the citrus root nematode, Tylenchulus semipenetrans belonging to the Tylenchulus genus, causes a detrimental effect on citrus plants. The genus, which belongs to the subfamily Tylenchulinae, leads to global yield losses in many crops, particularly in citrus species (O'Bannon, and Ford, 1978; Bello et al., 1986; Duncan et al., 1989; insera et al., 1994). Many citrus and citrus relatives from the Rutaceae family and several non-rutaceous crops, including grape, olive, and persimmon, have been reported to be the host of the genus (Inserra et al., 1988; Chawla et al., 1980; Shokoohi and Duncan, 2018). Tylenchulus semipenetrans was firstly reported from California in 1912; thereafter, its distribution was reported throughout the citrus-growing regions worldwide (Duncan, 2005). The three biotypes of T. semipenetrans as citrus, Mediterranean, and Poncirus have been recognized (Duncan et al., 1989; Inserra et al., 1980). Since T. semipenetrans is highly heterogeneous concerning its virulence to specific host genotypes, most of the commercial rootstocks have been become susceptible to T. semipenetrans (Mashela et al., 1992), ultimately leading to a disease called "slow decline of citrus" (Hannon, 1962; O'Bannon and Essar, 1994).

Among the plant-parasitic nematodes found in citrus orchards, T. semipenetrans was the highest rate of 81.2% (Abd-Elgawad et al., 2016). Trees infected with T. semipenetrans appear healthy, but their fruits shrink (Duncan, 2009). T. semipenetrans reproduced well at 25°C, and its population starts to decline when the temperature goes above 31°C and below 21°C (O'Bannon, et al., 1966). The nematode population is higher in the drip irrigation system than in the flood irrigation (Sorribas et al., 2000).

Accurate identification of PPN to the species level and beyond is needed as an initial step in designing effective control measures. As the species number of the genus Tylenchulus has been increased, identification based on morphological and morphometric features becomes more complicated and requires appropriate skills to discriminate the species from each other (Inserra et al., 1988; Kaplan, 1981; Stanton et al., 1997; Verdejo-Lucas and McKenry, 2004; Castillo and Vovlas, 2007). Recently, novel molecular diagnostic approaches have emerged and are being implemented to overcome misdiagnosis caused by conventional species description methods (Szalanski et al., 1997; Park et al., 2009; Subbotin et al., 2010). The mitochondrial cytochrome oxidase c subunit 1 (COI) gene, the internal transcribed spacer (ITS) region, large subunit (LSU), and small subunit (SSU) of ribosomal DNA are the most popular markers for population genetic and phylogeographic studies used in PPN identification (Subbotin et al., 2006; Madani et al., 2004; Smiley et al., 2008). The popularity of the COI gene region has gained even more preference since it appeared that the M1-M6 partition of the COI gene (hereafter referred to as the Folmer region) is an efficient identification tool for Metazoan species, turning it into the core fragment for DNA barcoding (Hebert et al., 2003). The ITS exhibits considerably high variations among nematode populations; thus, it is often used for nematode identification and phylogenetic studies at the species level (Inserra et al. 1988; Subbotin et al., 2005; Park et al., 2009; Subbotin et al., 2010; Smiley et al., 2008).

The intensity of T. semipenetrans on citrus species: Citrus reticulata, C. sinensis, C. limon, and C. parasidisi may differ (CABI, 2022). The citrus nematode significantly reduces lemon yields in Egypt (Hammam et al., 2021). In Turkey, T. semipenetrans was firstly reported from mandarin growing areas in Mersin and Adana provinces (Istiranca, 1940). Later, it was noted from other areas and became as a damaging pathogen for citrus and citrus relatives producing areas in Adana (Çukurova), Çanakkale (Gelibolu), and Rize provinces (Bodenheimer, 1958; Goffart, 1951). Currently, it is an important phytopathogenic nematode and responsible for serious damage in citrus and citrus relatives growing areas in Turkey. However, very little information is available on the genetic diversity of T. semipenetrans with different populations through the conventional diagnostic characteristics in Turkey (Toktay et al., 2005; Kasapoğlu et al., 2021).

To fill the informative gap, it is necessary to understand the population structures of T. semipenetrans; therefore, the major citrus and citrus relatives growing areas in Hatay, Osmaniye, Adana, and Mersin provinces were surveyed in order to (a) identify nematode populations by morphological and morphometrical methods as well as molecular techniques using of the ITS sequencing, (b) determine the phylogenetic relationships among these populations, (c) investigate host preference, (d) nematode density, (e) record economic-injury level (EIL) and economic threshold (ET).

A total of 73 samples containing soil and root parts were collected from citrus orchards in Hatay, Osmaniye, Adana, and Mersin provinces in the Mediterranean region of Turkey (Table 2). Sampling was conducted in the soil zone around the roots at a depth of 60 cm during September 2020. The sub-samples were pooled to form one representative composite sample of 1 kg of soil and 50 g of roots (Duncan et al., 1989) and stored in an incubator at 15-20°C to avoid decaying of nematodes specimens. Each sample taken from the roots/roots area of citrus host species was recorded. The number of nematode species was documented according to host species to determine the nematode host preferences. The Economic-injury level and Economic damage threshold are defined as the level at which the number of nematodes [2nd stage juveniles (J2s) + male nematodes] in 100 g of soil exceeds 3600 and 1600, respectively, for this nematode species (Garabedian et al., 1984; Erdem and Kaşkavalci, 2015) (Table 1).

Modified Baerman funnel method was used to extract infective J2s from 150 g soil per sample. Extracted J2s were transferred onto a 1 ml Hawksley counting slide then counted under a ZEISS Axio Lab. A1 light microscope (Carl Zeiss AG, Oberkochen, Germany) at 100× magnification.

A 10 g of roots were washed, cut into small pieces, and processed for nematode extraction by blender followed by centrifugal flotation. Roots were stained, and immature and mature females were observed on the root surfaces (Hooper et al., 2005). Later, female and egg masses were gently removed from roots under a ZEISS SteREO Discovery. V20 stereomicroscope (Carl Zeiss AG, Oberkochen, Germany) using a needle and collected with a thin brush in sterile deionized water and stored for species-level analysis.

Measurements of nematodes morphometrics: The extracted nematodes were fixed in a hot TAF (2 ml triethanolamine, 7 ml formaldehyde, and 91 ml distilled water) before being transferred to dehydrated glycerin (De Grisse, 1969). The J2s were placed in a little drop of dehydrated glycerin. The stained adult females were removed from the roots and placed in glycerin after being stained with diluted acid fuchsin lactophenol and cleared in a lactophenol solution. Character measurements for species description were carried out according to the method described by Insera et al (1988). Nematode measurements for 20 J2s and 20 females were performed using the microscope in the Nematology lab at Bolu Abant Izzet Baysal University (BAIBU). Females and J2s light micrographs were acquired with an automatic Zeiss camera system (High-Resolution AxioCam MRc 5, Carl Zeiss AG, Oberkochen, Germany) coupled to the Axio Lab. A1 compound microscopes.

Molecular characterization: For molecular analysis, mature female from each nematodes population were transferred to a glass slide and viewed using the ZEISS Axio Lab. A1 microscope in a drop of distilled water, then, they were rinsed in deionized water three times before being transferred to an Eppendorf tube containing 25 µl distilled water. After that, each Eppendorf tube was filled with a 10 µl drop of 1× PCR reaction buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 75 mM Tris-HCl (pH 8.8), 20 mM (NH₄)₂SO₄, and 0.01% Tween 20. A volume of 1 µl mercaptoethanol and 0.25 µl Proteinase K solution (> 600 mAU/ml, Qiagen GmbH, Hilden, Germany) were added to the mix. The tube was heated to 60°C for 15 min and then to 95°C for 5 min (Maafi etal., 2003). The lysate was stored at -20°C.

The universal primer set, AB28 (5'-CGTAACAAGGTAGCTGTAG-3') and TW81 (5'-TCCTCCGCTAAATGATATG-3') were used to amplify the ITS regions of T. semipenetrans populations. In a 50 µl PCR reaction mixture, 5 µl of the DNA lysate, 1 µl of each primer (10 µM), 4 µl of dNTPs (2 mM), 0.4 µl of Ampliqon TEMPase Hot Start DNA polymerase (Berntsen, Rdovre, Denmark), and 5 µl of 10× Ammonium buffer (Tris-HCl pH 8.5, (NH₄)₂S0₄, 15 mM MgCl₂, 1% Tween 20). The following parameters were used for PCR in a T100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA); an initial denaturation at 94°C for 12 min, followed by 35 cycles of 20 s at 94°C, 30 s at 55°C, 45 s at 72°C, and a final extension step of 10 min at 72°C. The amplification products were separated electrophoretically in a 1.6% agarose gel. A G: Box F3 gel documentation system was used to visualize and document the DNA banding profiles (Syngene, Cambridge, UK). The confirmed amplicons were sent to the Macrogen Inc. Sequencing Service (Seoul, Korea) for bidirectional sequencing.

Phylogenetic analysis: The newly acquired ITS sequences were read and edited checked manually, and nucleotide arrangements at ambiguous positions were clarified using both primer directions using MEGA X software (Kumar et al., 2018). Sequence alignment was constructed with their corresponding published gene sequences derived from the complete nucleotide library of the National Center for Biotechnology Information (NCBI)-GenBank using the online sequence alignment tool, MAFFT (https://mafft.cbrc.jp/alignment/server/large.html (Katoh et al., 2019). Nucleotide sequence comparisons were performed using the Basic Local Alignment Search Tool (BLAST) network services of NCBI with obtained sequences as a query to determine the closest available reference sequences. Phylogenetic analysis was done using PAUP (Phylogenetic Analysis Using Parsimony) version 4.0b10 (Swofford, 1998) for Maximum Likelihood analyses and visualized TREEFINDER version of March 2011 (Jobb, 2011) and MEGA X. The phylogenetic tree was rooted with the Tylenchulus musicola reference strain (JN112282). The resultant sequences were submitted to GenBank.

Statistical analysis was achieved according to Duncan's multiple range test to compare data using ANOVA by the SPSS program, and the significance value was P ≤ 0.05.

The citrus slow decline nematode, T. semipenetrans, is one of the main biotic stresses causes yield loss in citrus around the world (Bello et al., 1986; O'Bannon and Hutchinson, 1974; Duncan and Cohn, 1990). The present survey confirmed the occurrence of citrus nematode in Hatay, Osmaniye, Adana, and Mersin provinces in the Mediterranean region (Table 2). Since its discovery in 1912 in the roots of citrus trees in California and subsequently described by Cobb (1913) later, its incidence was reported from almost all over the world's major citrus-growing regions that the nematode was parasitizing citrus roots in Florida, Malta, Spain, Australia, and South America by 1914 (Cobb, 1914). In Turkey, T. semipenetrans was reported for the first time from mandarin growing areas in Mersin and Adana provinces (Istiranca, 1940). Later, it was reported from other areas and became recognized in citrus producing areas by Bodenheimer (1958) and Goffart (1951). The lowest density in the surveyed fields was obtained from Mersin province (80 J2s/150 g soil) in C. reticulata, while the highest density of 18260 J2s/150 g soil was reported from Adana province in C. reticulata (Table 2).

In the current study, T. semipenetrans was found in the determined in the 73 fields surveyed where citrus species (C. reticulata, C. paradisi, C. sinensis, and C. limon) are planted in Hatay, Osmaniye, Adana, and Mersin provinces in Turkey (Table 2). Similar to our findings, 90-95% citrus orchards in South California were found infested by this nematode (Thorne, 1961), 75% infestation rate was reported from citrus nurseries in India (Mani et al., 1988). Ahmad et al (2007) reported that 54.38% of the soil samples were infested with the citrus nematode in Sareodha in Pakistan. Tylenchulus semipenetrans cause damage in remarkable level on “banhazer” lemon (Citrus aurantifolia) as a population density of 2300 J2s/150 cm³soil cause 39.2 kg fruit loss/tree; 500 J2s/150 cm³soil cause 31.5 kg fruit loss/tree [13]. In this study, it is estimated that fruit losses of 21.9% of all citrus orchards in the Mediterranean region (Figure 5) may occur where more than 39 kg fruit /tree is lost (Abd-Elgawad et al., 2016).

Tylenchulus semipenetrans (nematode) numbers varied on the citrus host species: Citrus limon, C. paradise, C. reticulata, C. sinensis. C. reticulata and C. sinensis seem more susceptible as higher nematode numbers were extracted when compared to the citrus species of Citrus limon and C. paradise (Figure 3).

The average nematode density of the 73 surevyed fields as 3369.6 J2s/150 g soil in orchards (Table 2). The highest density of nematodes was 18260 J2s/150 g soil, obtained from sample Ts_H20 from Erzin-14 located in Hatay, followed by 18020 J2s/150 g soil, obtained from Ts_A3 sample from Kozan-3 in Adana province. The lowest density of nematodes was 80 J2s/150 g soil, extracted from Ts_M10 sample from Akdeniz-3 in Mersin followed 90 J2s/150 g soil, from Ts_M20 sample from Erdemli-7 in Mersin province. The number of female nematodes per gram of root varied depending on locations in the current study, which is due to various variables, including the population and infestation of citrus nematodes. Host variety and age, soil texture and moisture, pH, nitrogen status, temperature, and soil depth are all factors to consider. In addition, different races of nematodes have varying reproduction rates depending on rootstock (O'Bannon and Hutchinson, 1974; Iqbal et al., 2007). The nematode population number and spread are strongly influenced by tree age (Bello et al., 1986).

The mature female of T. semipenetrans was elongated anteriorly. The cuticle was smooth and without annulation. The Postvulval section cavity of body (PVSC), Postvulval section length of body (PVSL), Postvulval section width of body (PVSW) and Vulva excretory pore distance (VEPD) were measured as 4.48 µm, 32.42 µm, 13.24 µm, and 18.22 µm, respectively (Table 3).

The excretory pore is located at 67–80% of body length and at 10–25 μm to the vulva. Deirid was not noticeable. The intestine was with no apparent specializations. The reproductive system was monodelphic, prodelphic with reflexed ovaries. Mature females of T. semipenetrans occurring in Turkey were compared with those in the previous study (Table 3). The PVSC of Turkish poulation was longer than Park et al [22] (1.42-7.86 µm 1.31-5.24 µm vs 1.8-7.1 µm), and the PVSW was similar (11.52-16.60 µm vs 10.5-14.4 µm) with Rashidifard et al (2015). The cuticle thickness of T. semipenetrans of this study was larger than Inserra et al (1988) (4.08-10.55 µm vs 3.9-9.2 µm).

The body shape of J2s was almost straight after fixation. Cuticle had not formed of rings. The lateral field did not notice. The body and stylet length were measured 338.80-992.45 μm and 3.45- 6.96 μm, respectively (Table 3). The body length/maximum body-wide (a), the body length/oesophageal length (b) and the body length/tail length (c) was calculated 28.18- 98.48 μm, 3.65- 10.12 μm and 7.42- 27.12 μm, respectively (Table 3). The pharyngeal procorpus was cylindrical and longer than the postcorpus (isthmus + basal bulb). The metacorpus was swollen with a valve. The basal bulb was ovoid. Anus and rectum not visible. The hyaline portion of the posterior body has a few small fat globules. Tail with rounded terminus. The body length/maximum body-wide (a) was a high value compared with Park et al (2009), and the body length/oesophageal length (b) was a lower value compared with Rashidifard et al (2015). The body length/tail length (c) was similar to Inserra et al (1988) (6.9-27 µm vs 8-28 µm).

The cuticle outgrowths around the excretory pore are unique characteristics of the genus Tylenchulus (Inserra et al., 1988; Van den Berg and Spaull, 1982). A large extent of morphological variability among species populations was reported from economically important nematodes (Inserra et al., 1996; Handoo et al., 2005). It is important to know the variability of morphometric characters within a taxon to delimit species boundary (Duncan et al., 1989). In the previous studies Inserra et al. (1996) and Duncan et al. (1989), the essential criteria of nematode taxonomy, such as body length and width, were influenced by environmental conditions such as food availability and age of females in the population or unknown environmental conditions. In our study, some specimens showed that the mean value of cuticle thickness exceeded that of previous reports. Therefore, we need to examine more measurements and conduct additional experiments to verify potential environmental effects on the morphometric variation.

The sequencing and phylogenetic analysis of the ITS sequences were run for molecular diagnosis of T. semipenetrans populations obtained in surveyed areas. PCR amplification of the ITS region using primers AB28 and TW81 yielded a single PCR fragment of approximately 800 bp in size for all populations. DNA sequencing for the ITS region, including ITS1, 5.8S, ITS2, with partial 18S and 28S genes, displayed no length variations among the different populations examined. The consensus sequences were generated and finally used as BLAST queries against the GenBank nucleotide database. Seventy-three populations were identified as T. semipenetrans based on this sequence analysis. Nucleotide sequences of the ITS were deposited in GenBank under accession numbers listed in Table 2. The populations from Hatay, Osmaniye, Adana, and Mersin were compared with closely related Tylenchulus samples in GenBank.

Most of the T. semipenetrans populations had 99-100% similarity with the related T. semipenetrans samples recorded in the GenBank database. The phylogenetic tree of the T. semipenetrans populations from the provinces surveyed was shown in Figure 1. The phylogenetic tree was created from sequence alignments, which were rearranged globally with random replications. Minor intraspecific polymorphism was observed in the T. semipenetrans populations, which could be clustered in the one group within the phylogenetic tree, and representative populations from GenBank, supported by a moderate bootstrap value.

Regulatory concerns grow higher every year with the increased import of agricultural products. Developing a reliable and rapid nematode diagnostic method is an important step to detect of pests from infested areas. DNA sequence variation in the ITS region of the ribosomal DNA is useful to identify taxonomic groups among nematode groups. Heterogeneity, however, has been detected from many nematode species through comparative sequence analysis (Szalanski et al., 1997; Zheng et al., 2000; Han et al., 2006; Bae et al., 2008). In this study, ITS presented multiple bands exceeding the length of the unrestricted PCR products suggesting the coexistence of two ITS haplotypes in the same genome. These results may be related to the reproduction ability of T. semipenetrans. Obligatory mitotic (apomictic) parthenogenetic species, Meloidogyne arenaria, M. incognita, and M. javanica showed a high intraspecific genetic variation due to reticulate evolution (Hugall et al., 1994). To verify some characters of nematode evolution, we need to obtain and compare multiple sequences from amphimictic and parthenogenetic species. In the case of some species, the heterogeneity of the ITS region or expansion segments of the LSU gene did not exist in all populations of the same species. The emergence of heterogeneity within limited geographical populations of the same species may suggest the existence of a new local geographic subtype (Cherry et al., 1997; Zheng et al., 2000; Szalanski et al., 1997).

The density of many of these populations exceeded EIL and ET or approached the maximum threshold level for economic loss (Figure 2, 5). According to our knowledge, T. semipenetrans was the first comprehensive report in Hatay, Osmaniye, Adana, and Mersin provinces. The highest nematode number was determined in Hatay province which is located at the border of Syria, and the lowest number of nematodes was determined in Mersin province (Figure 4).

These results demonstrated the strongest analysis to date on the existence and distribution of T. semipenetrans in main citrus growing areas, thus serving as a basis for more specific resistance breeding, as well as other management practices. In conclusion, this study highlighted the diversity of T. semipenetrans in the Mediterranean region in Turkey, where, we found one species of Tylenchulus spp. that resemble their terrestrial counterparts molecularly. Additional studies are needed to explain this nematode species in citrus-growing areas in Turkey. Moreover, some further studies to describe T. semipenetrans populations in a detailed manner in the Turkish citrus-growing areas, and gene sources for resistance studies. Also, the sequence results obtained from this research presented the sequence data of the ITS region of T. semipenetrans found in Turkey. Several examinations of many populations collected from distant origins should be conducted to calculate the exact genetic variation within the species and develop molecular diagnostic techniques.

Author Contributions: Sample collections RB and Mİ; methodology, RB, Mİ and GÖ; Morphometric studies, MI; writing and original draft preparation RB and Mİ; phylogenetic GÖ; writing—review and editing, RB, SY, Mİ, GÖ, EY and AAD. All authors have read and agreed to the published version of the manuscript.

Disclosure statement: The authors report there are no competing interests to declare.

Funding: This research received no external funding.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available in the article.

Abd-Elgawad, M.M.M.; Koura, Faika, F.H.; Montasser, S.A. and Hammam, M.M.A., 2016. Distribution and losses of Tylenchulus semipenetrans in citrus orchards on reclaimed land in Egypt. Nematol. 18: 1141-1150. https://doi.org/10.1163/15685411-00003020.
Bae, C. H., A. L. Szalanski & R. T. Robbins, 2008. Molecular analysis of the lance nematode, Hoplolaimus spp., using the first internal transcribed spacer and the D1-D3 expansion segments of 28S ribosomal DNA. Journal of Nematology, 40(3): 201.
Bello, A., A. Navas & C. Belart, 1986. "Nematodes of citrus groves in the Spanish Levante, pp. 217-226". In: Integrated pest control in Citrus Groves (Eds: Cavalloro, R. & E. Di Martino). Balkena, Rotterdam, pp. 600.
Bodenheimer, F. S., 1958. The history of biology: an introduction. Dawson, London, pp. 18.
CABI, 2022. https://www.cabi.org/isc/datasheet/61059. (accessed on 01.02.2022)
Castillo, P. & N. Vovlas, 2007. Pratylenchus (Nematoda: Pratylenchidae): diagnosis, biology, pathogenicity and management. Brill, Leiden-Boston, pp. 530.
Chawla, M. L., G. J. Samathanam & S. B. Sharma, 1980. Occurrence of citrus nematode (Tylenchulus semipenetrans Cobb, 1913) on roots of mangosteen (Garcinia mangostana). Indian Journal of Nematology, 10(2): 240-242.
Cherry, T., A. L. Szalanski, T. C. Todd & T. O. Powers, 1997. The internal transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae). Journal of Nematology, 29(1): 23.
Cobb, N. A., 1913. New nematode genera found inhabiting fresh water and non-brackish soils. Journal of the Washington Academy of Sciences, 3(16): 432-444.
Cobb, N. A., 1914. Nematodes and Their Relationships. US Government Printing Office, NewYork, pp. 490.
De Grisse, A. T., 1969. Contribution to the morphology and the systematics of the Criconematidae (Taylor, 1936) Thorne, 1949. Contribution to the morphology and the systematics of the Criconematidae (Taylor, 1936) Thorne, 1949
Duncan, L. W. & E. Cohn, 1990. Nematode parasites of citrus, pp. 321-346". In: Plant parasitic nematodes in subtropical and tropical agriculture (Eds: Sikora, R.A., ‎D. Coyne, ‎J. Hallmann). CAB International, Wallingford, pp. 839.
Duncan, L. W., 2005. "Nematode parasites of citrus, pp. 437-466". In: Plant parasitic nematodes in subtropical and tropical agriculture (Eds: Sikora, R.A., ‎D. Coyne, ‎J. Hallmann). CAB International, Wallingford, pp. 839.
Duncan, L. W., J. J. Ferguson, R. A. Dunn & J. W. Noling, 1989. Application of Taylor's power law to sample statistics of Tylenchulus semipenetrans in Florida citrus. Journal of nematology, 21(4S): 707.
Duncan, L.W., 2009. Managing nematodes in citrus orchards. In: Ciancio A, Mukerji KG (eds) Integrated management of fruit crops and forest nematodes, Springer Science+Business Media B.V., pp 135-173 https: //doi.org/ 10.1007/ 978-1-4020-9858-1.
Erdem E., Kaşkavalci, G. 2015. The Determination of Distribution and Population Densities of Citrus Nematode [Tylenchulus semipenetrans (Cobb, 1913) (Tylenchida: Tylenchulidae)] in Satsuma Mandarin Growing Areas of Izmir Province . Ege Üniv. Ziraat Fak. Derg., 2015, 52 (3):269-276
Garabedian, S., Van Gundy, S. D., Mankau, R. and Radewald, J. D., 1984. Nematodes: In: Integrated Pest Management for Citrus. Univ. of California, Riverside, 129-131 pp.
Goffart, H., 1951. Nematoden der Kulturpflanzen Europas. Paul Parey, Berlin, pp.144.
Hammam, M., Abdel Gawad, M., Ruan, W., El-bahrawy, A. (2021). Management of Pests and Pathogens Affecting Citrus Yield in Egypt with Special Emphasis on Nematodes. Egyptian Journal of Agronematology, 20(2), 64-84. doi: 10.21608/ejaj.2021.183231
Han, H. R., A. Jeyaprakash, D. P. Weingartner & D. W. Dickson, 2006. Morphological and molecular biological characterization of Belonolaimus longicaudatus. Nematropica, 37-52.
Handoo, Z. A., A. M. Skantar, L. K. Carta & D. P. Schmitt, 2005. Morphological and molecular evaluation of a Meloidogyne hapla population damaging coffee (Coffea arabica) in Maui, Hawaii. Journal of Nematology, 37(2): 136.
Hannon, C. I., 1962. The occurrence and distribution of the citrus-root nematode, Tylenchulus semipenetrans Cobb. Florida. The Plant Disease Reporter, 46: 451-455.
Hebert PDN, Ratnasingham S, deWaard JR. 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London Series B-Biological Sciences. 270:S96–S99.
Hooper, D. U., F. S. Chapin Iii, J. J. Ewel, A. Hector, P. Inchausti, S. Lavorel & D. A. Wardle, 2005. Effects of biodiversity on ecosystem functioning: a consensus of current knowledge. Ecological monographs, 75(1): 3-35.
Hugall, A., C. Moritz, J. Stanton & D. Wolstenholme, 1994. Low, but strongly structured mitochondrial DNA diversity in root knot nematodes (Meloidogyne). Genetics, 136(3): 903-912.
Inserra, R. N., N. Vovlas, J. H. O'Bannon & R. Esser, 1988. Tylenchulus graminis n. sp. and T. palustris n. sp.(Tylenchulidae), from native flora of Florida, with notes on T. semipenetrans and T. furcus. Journal of Nematology, 20(2): 266.
Inserra, R. N., L. W. Duncan, N. Vovlas & P. A. A. Loof, 1996. Pratylenchus loosi from pasture grasses in central Florida. Nematologica, 42(2): 159-172.
Inserra, R. N., N. Vovlas & J. H. O'Bannon, 1980. A classification of Tylenchulus semipenetrans biotypes. Journal of Nematology, 12(4): 283.
Inserra, R., L. W. Duncan, J.H. O'bannon & S. A. Fuller, 1994. Citrus Nematode Biotypes and Resistance of Citrus Rootstocks In Florida. University of Florida Cooperative Extension Service, Florida, pp. 205.
Iqbal, M.A., T. Mukhtar & R. RAhmad, 2007. Effect of soil types on the movement and infection of citrus nematode, Tylenchulus semipenetrans. Pakistan Journal of Nematology, 25:69–72.
Istiranca, R., 1940. Narenciye Nematodu. Seyhan Ziraat Mücadele İstasyonu, Adana, pp. 399.
Jobb G. 2011. TREEFINDER version of March 2011. Munich. http://www. treefnder.de.
Kaplan, D. T., 1981. Characterization of citrus rootstock responses to Tylenchulus semipenetrans (Cobb). Journal of Nematology, 13(4): 492.
Kasapoğlu Uludamar, E. B. K., B. Çimen & H. Elekcioğlu, 2021. Doğu Akdeniz Bölgesi’nde doğa koşullarında bazı turunçgil anaçlarının Turunçgil nematodu’na [Tylenchulus semipenetrans (Cobb, 1913)(Tylenchida: Tylenchulidae)] karşı dayanıklılığının araştırılması. Bitki Koruma Bülteni, 61(1): 41-47.
Katoh K, Rozewicki J, Yamada KD., 2019. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 19;20(4):1160-1166. doi: 10.1093/bib/bbx108. PMID: 28968734; PMCID: PMC6781576.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol. 35(6):1547–1549.
Maafi, Z. T., S. Subbotin & M. Moens, 2003. Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology, 5(1): 99-111.
Madani, M., N. Vovlas, P. Castillo, S. A. Subbotin & M. Moens, 2004. Molecular characterization of cyst nematode Species (Heterodera spp.) from the Mediterranean Basin using RFLPs and sequences of ITS‐rDNA. Journal of Phytopathology, 152(4): 229-234.
Mani, A., V. Dakshinamurti & G. S. Reddy, 1988. Distribution of Tylenchulus semipenetrans and Meloidogyne javanica in commercial citrus nurseries in Andhra Pradesh. Indian Journal of Nematology, 18(2): 338-339.
Mashela, P., L. Duncan & R. McSorley, 1992. Salinity reduces resistance to Tylenchulus semipenetrans in citrus rootstocks. Nematropica, 7-12.
Ahmad, R., Khan, M. A., Mukhtar, T., & Javed, N. 2007. Infestation of citrus orchards by citrus nematode (Tylenchulus semipenetrans) and screening of some rootstocks for resistance or susceptibility. In Proceedings of the International Symposium on Prospects of Horticultural Industry in Pakistan. 28th to 30th March (pp. 165-169).
O'Bannon, J. H. & D. H. Hutchinson, 1974. "Nematode parasites of citrus, pp. 437-466". In: Plantparasitic nematodes in subtropical and tropical agriculture (Eds: Sikora, R.A., ‎D. Coyne, ‎J. Hallmann). CAB International, Wallingford, pp. 839.
O'Bannon, J. H. & H. W. Ford, 1978. Resistance in citrus rootstocks to Radopholus similis and Tylenchulus semipenetrans (Nematoda). Proceedings of the International Society of Citriculture, 2: 544-549.
O'Bannon, J. H. & R. P. Essar, 1994. Citrus declines caused by nematodes in florida I. (file:///C:/Users/Akife/Downloads/CH09700.PDF) (December 2021).
O'Bannon, J.H.; Reynolds, H.W. and Leathers, C.R. 1966. Effects of temperature on penetration, development, and reproduction of Tylenchulus semipenetrans. Nematologica, 12:483-487.
Park, B. Y., S. N. Park, J. K. Lee & C. H. Bae, 2009. Morphometric and genetic variability among Tylenchulus semipenetrans populations from citrus growing area in Korea. The Plant Pathology Journal, 25(3): 236-240.
Rashidifard, M., E. Shokoohi, A. Hoseinipour & S. Jamali, 2015. Distribution, morphology, seasonal dynamics, and molecular characterization of Tylenchulus semipenetrans from citrus orchards in southern Iran. Biologia, 70(6): 771-781.
Shokoohi, E. & L. W. Duncan, 2018. Nematode Parasites of Citrus. In: Plant parasitic nematodes in subtropical and tropical agriculture (Eds: Sikora, R.A., ‎D. Coyne, ‎J. Hallmann). CAB International, Wallingford, pp. 839.
Smiley, R. W., G. P. Yan & Z. A. Handoo, 2008. First record of the cyst nematode Heterodera filipjevi on wheat in Oregon. Plant Disease, 92(7): 1136-1136.
Sorribas, F.J.; Verdejo-Lucas, S.; Forner, J.B.; Alcaide, A.; Pons, J. and Ornat, C. (2000). Seasonality of Tylenchulus semipenetrans Cobb and Pasteuria sp. in citrus orchards in Spain. Supplement to J. Nematol, 32(4S):622–632.
Stanton, J., A. Jugall & C. Moritz, 1997. Nucleotide polymorphisms and an improved PCR-based mtDNA diagnostic for parthenogenetic root-knot nematodes (Meloidogyne spp.). Fundamental and Applied Nematology, 20(3): 261-268.
Subbotin, S. A., D. Sturhan, V. N. Chizhov, N. Vovlas & J. G. Baldwin, 2006. Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology, 8(3): 455-474.
Subbotin, S. A., M. Madani, E. Krall, D. Sturhan & M. Moens, 2005. Molecular diagnostics, taxonomy, and phylogeny of the stem nematode Ditylenchus dipsaci species complex based on the sequences of the internal transcribed spacer-rDNA. Phytopathology, 95(11): 1308-1315.
Subbotin, S. A., M. Mundo-Ocampo & J. Baldwin, 2010. "Systematics Of The Heteroderinae, pp. 101-106". In: Systematics of Cyst Nematodes (Nematoda: Heteroderinae) (Eds: Subbotin, A.S., ‎M. Mundo-Ocampo, ‎J.G. Baldwin). Brill, NewYork, pp. 524.
Swofford, D. L. 1998. PAUP*. Phylogenetic analysis using parsi-mony (*and Other Methods). Vers. 4. Sinauer, Sunde land, MA
Szalanski, A. L., D. D. Sui, T. S. Harris & T. O. Powers, 1997. Identification of cyst nematodes of agronomic and regulatory concern with PCR-RFLP of ITS1. Journal of nematology, 29(3): 255.
Thorne, G., 1961. Principle of Nematology. McGraw-Hill Book Company, Inc.., New York, pp. 553.
Toktay, H. , Elekçioğlu, İ. , Özarslan, A. 2005. "Determination of Citrus nematode (<em>Tylenchulus semipenetrans</em> Cobb.) biotypes in East Mediterranean region of Turkey.". Plant Protection Bulletin 45: 9-16
Van den Berg, E. & V. W. Spaull, 1982. Longidoroides species from sugar cane in Natal (Nematoda: Longidoridae). Phytophylactica, 14(4): 179-194.
Verdejo-Lucas, S. & M. V. McKenry, 2004. Management of the citrus nematode, Tylenchulus semipenetrans. Journal of Nematology, 36(4): 424.
Zheng, J., S. A. Subboth, L. Waeyenberge & M. Moens, 2000. Molecular zcharacterization of Chinese Heterodera glycines and H. avenae populations based on. Russian journal of Nematology, 8(2): 109-113.

Table 1. The classification of the population densities of Tylenchulus semipenetrans[36, 37].

Damage Level Scale Value	Damage Level	Population Density /100 g Soil
1	Does not cause economic damage	<1600
2	Economic damage threshold (ET)	>1600
3	Economic-injury level (EIL)	>3600

Table 2. The information of Tylenchulus semipenetrans populations obtained in the current study

Sample ID	Location	Host	Density (J2s/150g soil)	Accession Number
Ts_H3	Hatay, Erzin-3	C. reticulata	16240	OL069268
Ts_H4	Hatay, Erzin-4	C. reticulata	11230	OL069269
Ts_H5	Hatay, Erzin-5	C. reticulata	150	OL069270
Ts_H6	Hatay, Erzin-6	C. reticulata	6280	OL069271
Ts_H7	Hatay, Erzin-7	C. reticulata	500	OL069272
Ts_H8	Hatay, Erzin-8	C. reticulata	160	OL069273
Ts_H9	Hatay, Dörtyol-1	C. reticulata	460	OL069274
Ts_H11	Hatay, Dörtyol-3	C. reticulata	180	OL069275
Ts_H12	Hatay, Dörtyol-4	C. reticulata	810	OL069276
Ts_H13	Hatay, Dörtyol-5	C. sinensis	1480	OL069277
Ts_H14	Hatay, Dörtyol-6	C. sinensis	550	OL069278
Ts_H15	Hatay, Erzin-9	C. sinensis	8720	OL069279
Ts_H16	Hatay, Erzin-10	C. sinensis	12140	OL069280
Ts_H17	Hatay, Erzin-11	C. reticulata	4520	OL069281
Ts_H18	Hatay, Erzin-12	C. paradisi	680	OL069282
Ts_H19	Hatay, Erzin-13	C. paradisi	650	OL069283
Ts_H20	Hatay, Erzin-14	C. sinensis	18260	OL069284
Ts_M1	Mersin, Tarsus-1	C. sinensis	440	OL069285
Ts_M2	Mersin, Tarsus-2	C. limon	480	OL069286
Ts_M3	Mersin, Tarsus-3	C. sinensis	230	OL069287
Ts_M4	Mersin, Tarsus-4	C. sinensis	9870	OL069288
Ts_M5	Mersin, Tarsus-5	C. limon	530	OL069289
Ts_M6	Mersin, Tarsus-6	C. limon	9500	OL069290
Ts_M7	Mersin, Tarsus-7	C. sinensis	220	OL069291
Ts_M8	Mersin, Akdeniz-1	C. limon	350	OL069292
Ts_M9	Mersin, Akdeniz-2	C. reticulata	980	OL069293
Ts_M10	Mersin, Akdeniz-3	C. reticulata	80	OL069294
Ts_M11	Mersin, Mezitli-1	C. limon	280	OL069295
Ts_M13	Mersin, Mezitli-3	C. limon	190	OL069296
Ts_M15	Mersin, Erdemli-2	C. sinensis	280	OL069297
Ts_M16	Mersin, Erdemli-3	C. limon	680	OL069298
Ts_M17	Mersin, Erdemli-4	C. limon	610	OL069299
Ts_M18	Mersin, Erdemli-5	C. sinensis	3260	OL069300
Ts_M19	Mersin, Erdemli-6	C. limon	180	OL069301
Ts_M20	Mersin, Erdemli-7	C. limon	90	OL069302
Ts_O1	Osmaniye, Toprakkale-1	C. sinensis	980	OL069303
Ts_O2	Osmaniye, Toprakkale-2	C. reticulata	210	OL069304
Ts_O3	Osmaniye, Toprakkale-3	C. reticulata	14260	OL069305
Ts_O4	Osmaniye, Kadirli-1	C. reticulata	3120	OL069306
Ts_O5	Osmaniye, Kadirli-2	C. reticulata	470	OL069307
Ts_O6	Osmaniye, Kadirli-3	C. paradisi	630	OL069308
Ts_O8	Osmaniye, Kadirli-5	C. reticulata	630	OL069309
Ts_O9	Osmaniye, Kadirli-6	C. reticulata	820	OL069310
Ts_O10	Osmaniye, Sumbas-1	C. reticulata	1560	OL069311
Ts_O11	Osmaniye, Sumbas-2	C. sinensis	670	OL069312
Ts_O12	Osmaniye, Sumbas-3	C. reticulata	450	OL069313
Ts_O13	Osmaniye, Sumbas-4	C. reticulata	260	OL069314
Ts_O14	Osmaniye, Sumbas-5	C. reticulata	10260	OL069315
Ts_O15	Osmaniye, Sumbas-6	C. sinensis	840	OL069316
Ts_O16	Osmaniye, Sumbas-7	C. reticulata	1750	OL069317
Ts_O17	Osmaniye, Sumbas-8	C. reticulata	3860	OL069318
Ts_O18	Osmaniye, Sumbas-9	C. reticulata	3010	OL069319
Ts_O19	Osmaniye, Sumbas-10	C. reticulata	14020	OL069320
Ts_O20	Osmaniye, Sumbas-11	C. reticulata	380	OL069321
Ts_A1	Adana, Kozan-1	C. reticulata	5820	OL069322
Ts_A2	Adana, Kozan-2	C. reticulata	1510	OL069323
Ts_A3	Adana, Kozan-3	C. reticulata	18020	OL069324
Ts_A5	Adana, Kozan-5	C. sinensis	660	OL069325
Ts_A6	Adana, Kozan-6	C. limon	480	OL069326
Ts_A7	Adana, Kozan-7	C. reticulata	4160	OL069327
Ts_A8	Adana, Kozan-8	C. reticulata	420	OL069328
Ts_A9	Adana, Kozan-9	C. reticulata	7210	OL069329
Ts_A10	Adana, Kozan-10	C. reticulata	6260	OL069330
Ts_A11	Adana, Kozan-11	C. reticulata	4110	OL069331
Ts_A12	Adana, Seyhan-1	C. sinensis	470	OL069332
Ts_A13	Adana, Seyhan-2	C. sinensis	280	OL069333
Ts_A14	Adana, Seyhan-3	C. reticulata	1160	OL069334
Ts_A15	Adana, Seyhan-4	C. sinensis	1860	OL069335
Ts_A16	Adana, Seyhan-5	C. paradisi	1210	OL069336
Ts_A17	Adana, Seyhan-6	C. sinensis	6250	OL069337
Ts_A18	Adana, Seyhan-7	C. sinensis	3860	OL069338
Ts_A19	Adana, Seyhan-8	C. sinensis	660	OL069339
Ts_A20	Adana, Yüreğir-1	C. reticulata	540	OL069340

Table 3. Measurements of Tylechulus semipenetrans mature females and second-stage juveniles (J2s) (n=20)

Characters	Range (µm)	Mean+SD	Coefficient of variation (CV) (%)
Females
Body width at vulva (BWV) (µm)	14.8-34.40	25.80±3.44	1.22
Postvulval section cavity of body (PVSC) (µm)	1.42-7.86	4.48±1.45	2.05
Postvulval section length of body (PVSL) (µm)	24.60-48.50	32.42±2.28	1.03
Postvulval section width of body (PVSW) (µm)	11.52-16.60	13.24±1.18	0.85
Vulva excretory pore distance (VEPD) (µm)	12.60-28.75	18.22±3.45	1.24
Cuticle thickness (CT) (µm)	4.08-10.55	7.27±1.2	1.42
Second stage juveniles (J2s)
Body length (L) (µm)	338.80-992.45	595.45±82.80	30.22
Stylet length (µm)	3.45-6.96	4.96±0.85	17.24
Body length/maximum body wide (a)	28.18-98.48	68.28±10.25	40.28
Body length/oesophageal length (b)	3.65-10.12	6.75±2.27	30.44

Download PDF

Version 1

posted

You are reading this latest preprint version

Nematode-citrus plant interactions: host preference, damage rate and molecular characterization of Citrus root nematode Tylenchulus semipenetrans

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

3. Results And Discussion

Declarations

References

Tables

Status:

Version 1