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 cm3 soil cause 39.2 kg fruit loss/tree; 500 J2s/150 cm3 soil cause 31.5 kg fruit loss/tree . 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  (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.