Community Analysis of Nematodes Associated with Banana, Identication of root knot nematode and Evaluation the Susceptibility of Some Cultivars to Infection

Background Plant-parasitic nematodes are extremely dangerous pests in a variety of economically important crops. The purpose of this study was a survey of all nematode species existing in banana from three sites in Assiut Governorate, Egypt and to characterize the most common species by morphological, morphometric and molecular techniques (PCR with species-specic primers). Then, study of resistance or sensitivity of some banana cultivars to root-knot nematodes. Methods and Results Four nematodes, Meloidogyne, Rotylenchulus reniformis, Helicotylenchus and Pratylenchus were isolated and identied from soil and root samples collected from banana plants. Most frequently occurring of plant parasitic nematode species in banana was Meloidogyne. Former research found differences in species and in resistance to root-knot nematodes among the examined plant cultivars. Identication of Root-knot nematodes by Characterize of morphometric, molecularly, morphological isolate of Meloidogyne related to banana plants. The results revealed that the identied nematode species, Meloidogyne javanica, is the most common plant-parasitic nematodes in all locations. Data on the susceptibility of the tested banana cultivars to M. javanica revealed that Grand Naine was highly susceptible (HS) however, Magraby was susceptible (S) but Williams and Hindi cultivars were moderately resistant (MR). Conclusions we concluded that a survey revealed the signicant prevalence of Meloidogyne javanica, the most important nematodes on banana in Assiut. The morphometric, morphological, and molecular identication were harmonic with one another. In addition to the host response of certain banana cultivars, to M. javanica that resistance is of signicance and can be helpful to incorporate through planning control measures for root- knot nematodes.


Introduction
Banana (Musa sp.) is one of the world's most economic tropical fruit crops. Bananas thrive in a wide range of soil conditions, also an important source of carbohydrates, ber, proteins, vitamins, and minerals. Its cultivation area in Egypt reached approximately 447299 ha, with an average yield of 1359297 tons/ha. [1]. Nematodes are one of the world's most signi cant limitations on banana production, with 146 species recorded in 43 genera. A most dangerous nematode species are that, devastate the primary roots, causing the anchorage system to fail and the plant to fall over [2]. The major nematode pests of banana include burrowing nematode, Radopholus similis; root knot nematodes, Meloidogyne arenaria, M. incognita and M. javanica; spiral nematodes, Helicotylenchus multicinctus; and H. dihystera; root lesion nematodes, Pratylenchus coffeae, P. reniformiya P. brachyurus and P. goodeyi,; and reniform nematode, Rotylenchulus reniformis [3,4]. The perineal pattern is predominantly uncertain when used alone to make diagnostic deduction, but when used as an integral device in conjunction with morphometric characterization or molecular techniques, it is substantial for screening the morphological matchmaking of the identi cation [5]. So that, molecular diagnostics of Meloidogyne sp. have been research as a surrogate. PCR-based disclosure methods, such as species speci c or sequence characterized ampli cation region (SCAR) primers, have been advanced and vastly used for nematode identi cation [6]. SCAR markers are preferred over RAPD markers because they detect only a single locus and are more speci c. Furthermore, their PCR ampli cation is low sensible to reaction constraints and thus more reproducible. Nematode relationships with bananas, including damage, depend on environmental conditions, susceptibility of the host and pathogenicity of the nematode considered [7]. Due to recent concerns about pesticide contamination of the environment, the search for both plan resistance and/or tolerance to plant-parasitic nematodes of bananas is now a major challenge to supplement the existing IPM approach, gaining new interest and involving many research teams [8].
The objective of the present study was a survey of all nematode species existing in banana from three sites (Assiut Center, Al-Fath, Sahel Saleem) in Assiut Governorate, Egypt and to characterize the most common species by morphological, morphometric and molecular techniques (PCR with species-speci c primers). Then, study of resistance or sensitivity of some banana cultivars to root-knot nematodes.

Materials And Methods
Plant-parasitic nematodes associated with banana plants in Assiut Governorate: Three sites (Assiut, Sahel-Selim and El-Fath) were selected for the nematode screening based on contrasting soil types and the availability of diverse Musa species under commercial cultivation. All sites had been commercial banana have favorable climatic conditions for Musa agricultural production.

Sampling and Collection:
Two to four banana plants were sampled for each accession at each site preferentially from recently owering plants. Root and soil samples were collected from an approximately 30 cm x 30 cm area on two sides of each plant and placed into a plastic bag properly identi ed with the date, location, and cultivar name. All samples were transported in insulated boxes to protect samples from direct sunlight and temperature uctuations. Samples were stored at 4°C until processing for nematode extraction.

Nematode extraction:
Cobb's decanting and sieving method [9] was used to extract the nematodes from a 100g combined soil sample, accompanied by amended Baermann procedure [10].
Taking 20g of pencil-thick banana feeder roots, gently wash them under tap water, cut them into little bits of 2-3 cm in length, split them longitudinally, and place them on double-layered facial tissue paper, then follow the amended Baermann procedure [10]. Keep this society to sit unmoved for 48 hours before collecting nematode suspension to examine under a microscope. After differential staining with NaOCl -acid fuchsin technique, the root samples were analyzed using a stereoscopic microscope (Carl Zeiss-Stemi 2000C) [11].

Estimate of nematode populations:
Flask containing stable nematode suspension was taking in a measuring cylinder to measurement the total quantity of nematode suspension acquired from 100g of soil. Nematodes stayed in the constant suspension were calculated under binocular microscope using multichambered counting plate. The nematode suspension from each location was spotted and mean of three aliquots was taken for calculating the population density per 100g of soil.

Processing of nematodes
The xed nematode samples from each population were analyzed when using glycerol-ethanol method for morphological and morphometric studies [12]. The processed specimens were permanently placed in pure anhydrous glycerol. A small drop of glycerol was placed in the center of a clean glass slide (Borosil brand) that measured 76 mm 26 mm 1.25 mm. Approximately 8-10 processed nematode specimens were picked up and located in the middle of a glycerol drop with their heads leading in the same tren, ensuring that they were resting on the surface of the glass slide and not hovering on the surface of the drop. A microscopic cover glass measuring 18 mm was placed over the specimen and sealed with a para n wax ring [13].
Identi cation of Plant parasitic nematodes from different banana growing areas: Important morphological and morphometric features of taxonomic signi cance have been studied in detail for each population of Plant parasitic nematodes obtained from different banana growing areas. The parameters used to characterize nematode species were developed initially by [14] and added to, modi ed and amended by [15,16] and others.
Identi cation of root-knot nematodes: Morphometrical characterization: Morphometric dimensions of Meloidogyne were speci ed on ten individual J2 from three center. J2 was tentatively mounted in water on glass slides before being spotted and measured at 100 magni cations with a compound light microscope (OMAX 40X-2000X digital binocular biological compound microscope) linked to a computer working Scope-Image-9.0 Professional Imaging software. The optical microscope was used to measure ve morphometric variables (stylet length, tail length, body length, hyaline terminus length, and the distance between the stylet base and the dorsal esophageal gland ori ce (DEGO)).

Morphological characterization:
Sections of infected roots should be immersed in 0.9% NaCl. Using a dissecting microscope, separate females from roots by needle and a scalpel and transfer the females to a petri dish with a small drop of 45% lactic acid. Push a female body out of a drop in a small isthmus of lactic acid solution, so that surface tension holds it in place. Insert the razor blade fragment into the slide and use a paper cutter to cut off the nematode's posterior. Using a dissecting needle, gently remove body tissue from the posterior section. In a small drop of glycerin, place the perineal pattern on a microscope slide. The internal surface of the cuticle should place against the glass then, cover slip placed on the glycerin drop. [17].

Molecular characterization:
DNA extraction: The CTAB (cetyltrimethylammonium bromide) method was used to extract DNA from nematode isolates [18] with some modi cations. Many adult females gained from each isolate were frozen in liquid nitrogen then, crushed using a suitable pestle and mortar. 600μl of CTAB extraction buffer was added to each sample and the mixture was then transferred to 1.5 ml Eppendorf tube. A volume of 50μl βmercaptoethanol was added and all tubes were well vortexed for 15 sec and then incubated for about 40 min at 65 o C in a water bath. After incubation, the tubes were kept at room temperature for 5-10 min, and 600μl chloroform: isoamyl alcohol solution (24:1 v/v) was then added to each tube, and the solution was gently mixed. The tubes were then subjected to a centrifugation (8,000 rpm at 4° C for 15 min). After the centrifugation, approximately 500μl of the upper aqueous phase (without any solid material) was transferred to a new 1.5 tube and an equal volume (500 μl) of cold isopropanol was added to each tube.
The tubes were then slowly inverted several times and stored in the refrigerator overnight. A centrifugation (13,000 rpm at 4° C for 10min) was performed for the tubes. After the centrifugation, the supernatant was discarded and the DNA pellet was then washed by adding 1 ml of 70% cold ethanol, and a centrifugation (13,000 rpm at 4° C for 5 min) was performed. The tubes were kept at room temperature to allow the DNA pellet to air-dry (approximately 15 min). The dried DNA pellet was then resuspended in 100 μl TE buffer. DNA concentration (μg/ml) was determined for each sample by using spectrophotometer, and required dilutions were then performed to be used later for PCR.

Species-speci c PCR assay:
A species-speci c SCAR primer collection Table 1, selected from previous studies [19] as a speci c marker for Meloidogyne javanica, namely Fjav/Rjav, was used to con rm morphological identi cation of nematode isolates. Ampli cations were carried out in 25μl reaction mixtures containing 5-10 ng of genomic DNA, 1X PCR buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 0.8 μM of each primer, and 1 U Taq DNA-polymerase and using the following PCR software in a Senso Quest Lab Cycler (SensoQuest GmbH, Göttingen, Germany): 5 minutes at 95° C, then 35 intervals of 1 minute at 94° C, 1 minute at 58° C, and 1 minute at 72° C, followed by one nal extension period at 72° C for 10 minutes. PCR products were separated on a 1.5 percent agarose gel stained with ethidium bromide in 0.5 X TBE buffer using a horizontal gel electrophoresis unit. The size of each ampli ed DNA fragment was determined using a DNA ladder. The gel was run for about an hour at a constant voltage of almost 80 V, and then photographed using a gel documentation device under UV light. For each SCAR marker, the same band with the predicted size was then detected separately. Table 1 SCAR primers were used to identify M. javanica at the molecular level.

Sequence (5'-3') Reference
Fjav/Rjav 670 GGTGCGCGATTGAACTGAGC CAGGCCCTTCAGTGGAACTATAC [19] Susceptibility of certain banana cultivars to M. javanica: This experiment was conducted at the greenhouse of Plant Pathology Department, faculty of Agriculture, Assiut University. Nematode free seedlings, of four rootstocks; (Hindi, Magraby, Williams and Grand Naine) were used for evaluating their susceptibility to the Root-Knot nematode, M. javanica. They were obtained from the Horticultural research Centre, Giza. Three -month old seedlings of each cultivar were grown in 40cm pots lled with sterilized sandy-clay soil (4 Kg soil to each pot).
Inoculation of root-knot nematode was taken from the stock culture and nematode eggs were extracted from the roots using sodium hypochlorite 0.5 % solution [20]. After 25 days of seedling transplanting inocula was added as 5000 J2 per plant according to [21] with modify. The inocula were added in three holes around plant roots with micropipette and pots were watered daily and fertilized every week with 1 g /plant of NPK salt (1:1:1).
Data were recorded after 90 days from nematode inoculation. Nematode population was estimated using Baermann pan technique [22] as previously mentioned.

Results
In the sampled areas, phytonematodes occurred in polyspeci c communities comprising a mixture of Meloidogyne, Pratylenchus, Helicotylenchus, and Rotylenchulus reniformis. The root-knot nematode (Meloidogyne) was dominant in all localities (Assiut Center, Sahel-Selim and El-Fath). Higher frequencies were observed in Sahel-Selim and El-Fath, with frequencies of 1150 and 1125 J2/250g soil respectively ( Table 2). Rotylenchulus reniformis presented a low frequency 125 J2/250g soil in Sahel-Selim Table 2. Second-stage juveniles were vermiform and slender ranged from 400-550 μm in length and a head that was not offset from the body. Stylet knobs transversely elongate and are offset from the stylet shaft, stylet length ranged from 9.6-12.4 μm. The distance between the dorsal esophageal gland and the base of the stylet was 3-4 m. Tail length was 50-62.2 μm with rounded tip. The hyaline tail length ranged from 10.2 to 18.4 μm, with a long slender tapering tail and a delicately curved tail tip, which corresponded to the characterization set for M. javanica by [24,25]. Morphometric performed on J2 are reported in Table 3.

Perineal Pattern Morphology:
When comparing to previous reports, assessment of the perineal pattern's morphology of adult females from three localities, selected by hand from infected banana roots, revealed brow model of M. javanica [24]. M. javanica was dominant in the three localities. The perineal patterns of M. javanica are unrivaled because they consist of side ridges that part the dorsal and ventral lines. In generally, the ridges run the entire width of the pattern, but progressively die out near the tail end. The dorsal arch is faint and rounded to high and squarish and often contains a whorl in the tail terminal area. The striae are sleek to little adverse, and several striae may curvature across the vulval edges Fig. 1.

Molecular identi cation of Meloidogyne javanica:
One species-speci c SCAR primer pairs, namely Fjav/Rjav were used for molecular diagnosis of nematode isolates to further con rm species identi cation. The PCR assay was performed on one sample from each location. The three nematode isolates clearly ampli ed the expected speci c DNA fragment of 670 bp Fig. 2 which con rms the identi cation of M. javanica as recorded by [19].

Susceptibility of certain banana cultivars to M. javanica
Data on the susceptibility of the tested banana cultivars to M. javanica revealed that Grand Naine was highly susceptible (HS), Magraby was susceptible (S), but Williams and Hindi were moderately resistant (MR) Table 3.
The number of galls and egg masses of root-knot nematode were counted. Plants were graded based on the number of egg masses found on their roots.

Discussion
This study identi ed the most frequent and abundant nematode species isolated from soil and banana roots from two long-term banana plantings in Assiut Governorate. Meloidogyne, and Pratylenchus are known to cause the serious economic damage to banana plantations, it is agree with [7]. Meloidogyne were, in general, the most abundant phytonematodes recovered from soil and root samples.
These results relative to root-knot nematode population are in accordance with those reported by several investigators as an important nematode pest attacking banana in different countries, [26] in West and Central Africa, [27] in Crete, [3] in Hawaiian Islands, [28] in West Bengal, [29] in Rusitu Valley, [30] in Egypt, [31] in Republic of Congo, [32] in Ethiopia, [33] in South Africa, [34] in Turkey, [35] in Lasbela Balochistan and [36] in Southern Florida.
Morphometric and morphological studies necessitate a signi cant amount of endeavor and are not always simple, attributed to the prevalence of intra-speci c variations. As the previously reported, the morphometric values overlap, and the morphology of the perineal pattern, while extra helpful but, remains indecisive due to individual variability, the assorted practice of those describing the patterns, and the increased numbering of species. The combination of morphology and morphometric may provide a small hint toward species identi cation. This result is agreement with [37,38,39,40]. M. javanica is the most frequent Meloidogyne sp. found in tropical and subtropical areas [41], such as Egypt, where annual temperatures range between 17-32˚C. It is important to note that, while PCR is quick, simple, and capable of determining species identity regardless of developmental phase and from tiny portions of tissue.
According to this study, Because of intraspeci c variability and species closeness, its dependability is uncertain. Thus, morphology, morphometric, and molecular analysis work in tandem to provide more accurate and reliable identi cation. The PCR examination for the nematode isolates with the speci c SCAR primer Fjav/Rjav clearly produces a speci c DNA piece of 670 bp (Fig. 2) which con rms the identi cation of M. javanica such results were in harmony with those [19,42,43,44]. SCAR markers have been to a large degree used in molecular identity of root-knot nematodes, both to prove morphological identi cations and to set apart unknown isolates in genomic analysis [19,45,42,46,43].
Susceptibility of four banana cultivars (Hindi, Magraby, Williams and Grand Naine) to M. javanica. The glasshouse experiment indicated some differences in the response between the cultivars. 'Grand Nain' was most sensitive to M. javanica, whereas 'Williams' allowed less reproduction of this nematode. Such results agree with those reported by [47,48,49]. However, disagree with reported by [33].

Conclusions
From the results we concluded that a survey revealed the signi cant prevalence of Meloidogyne javanica, the most important nematodes on banana in Assiut. As, these species are known to cause severe damage and yield losses throughout most banana growing areas in the tropical and subtropical regions of the world. The morphometric, morphological, and molecular identi cation were harmonic with one another, implying that molecular analysis of root-knot nematodes using SCAR markers could be used as a supplement to morphometric and morphological identi cation. In addition to the host response of certain banana cultivars, to M. javanica that resistance is of signi cance and can be helpful to incorporate through planning control measures for root-knot nematodes.

Declarations
Author contributions Radwa G. Mostafa: Methodology, Validation, Formal analysis, Investigation, Writing-original draft. Aida M. El-Zawahry: Supervision, Conceptualization, make substantial contributions to conception and design, Resources, Validation, Writing-review& editing. Ashraf E. M. Khalil: Supervision, Conceptualization, Resources, make substantial contributions to conception and design. Ameer E. Elfarash: participate in drafting the article or revising it critically for important intellectual content. Ali D. A. Allam: Supervision, Conceptualization, Resources. All authors give con rmation of their consent to participate in this manuscript sent for publication. All authors also give nal approval to the submitted version and any revised versions Data availability The data supporting the study's ndings are available upon request from the corresponding author. Due to privacy and ethical concerns, the data is not publicly available.
Con ict of interest: Figure 1 Perineal pattern of M. javanica.