Biodiversity and Community Analysis of Plant-Parasitic and Free-Living Nematodes Associated with Maize and Other Rotational Crops from Punjab, Pakistan

Maize (Zea mays L.) is one of Pakistan’s essential staple food crops. Plant-parasitic nematodes (PPNs) are a significant restraint in maize production. However, free-living nematodes (FLNs) provide crucial ecological functions such as suppressing pests and nutrient mineralization. This study aimed to assess the community analysis of plant-parasitic and free-living nematodes associated with maize and other rotational crops (those cultivated in sequence with the maize in the same field) from Punjab, Pakistan. The occurrence percentage was observed per 500 g soil for each nematode genus. The present study revealed that 24 species of plant-parasitic and free-living nematodes were identified from maize crops and other rotational crops from 16 localities through Punjab, Pakistan. Nematode communities were analyzed by absolute frequency, relative frequency, relative density, and prominence value, while cluster analysis was based on the presence or absence of nematode in different localities. The overall proportion of plant-parasitic nematodes was 35%, while free-living soil nematodes recovered 65%, out of 210 samples of maize and other rotational crops. Several major genera of plant-parasitic nematodes were reported during the present study viz., Ditylenchus, Filenchus, Helicotylenchus, Hemicriconemoides, Heterodera, Hoplolaimus, Malenchus, Pratylenchus, Psilenchus, Rotylenchulus, Seinura, Telotylenchus, Tylenchorhynchus, and Xiphinema Community relationship revealed the overall dominance of Heterodera zeae, with the highest incidence (55.71%) followed by Tylenchorhynchus elegans (33.33%) and Helicotylenchus certus (24.76%). The results provide valuable information on the community structure of nematodes in maize and other rotational crops of maize in Punjab, Pakistan. Moreover, this data can be used as a preventive measure before PPN incidence results in greater losses on maize.


Introduction
Maize (Zea mays L.) is one of the important cereal crops grown worldwide [1]. It is the most preferred and popular crop in semi-arid and arid regions of the world [2]. It is a good source of food consumed throughout the world and an important feed component for livestock [3]. Maize is high-yielding, easy to digest, and cheaper than any other cereals [4]. Every part of this plant has an economic value: grain, cob, stalk, leaves, and tassel are used for producing a variety of food and non-food products [5]. Maize is a main source of starch (72%), proteins (10%), fiber (5.8%), vitamins A and B (3-5%), sugar (3.0%), oil (4.8%), and ash (1.7%) [6]. Moreover, 100 g of fresh grains contains 361 calories of energy, 74.4 g carbohydrate, 9.4 g protein, 4.3 g fat, 1.8 g fibres, 1.3 g ash, 9 mg calcium, 290 mg phosphorus, 2.5 mg iron, and 140 mg vitamin [7]. Maize is rich in starch, so it is used as raw material for several products, i.e., glucose, lactic acid, alcohol, plastic, starch, rayon, Pull Bagar, Shamsabad, Sheikhupura, and 123 EB Pakpattan Canal were the locations surveyed ( Figure 1). A total of 210 soil and roots samples of maize and other rotational crops (potato, sorghum, wheat, tobacco, and green chilli) were obtained. The samples were collected during the spring season (March-April). Nematode distribution is rarely uniform or steady, and changes can happen quickly. The distribution of nematodes is usually uneven. Thus, the survey field that will be sampled was divided into subdivisions. Then, the effective sampling map was constructed. Large and small survey fields were sampled in a systematic, zigzag way. The main aim of the survey was to observe the prevalence and occurrence of nematode-related diseases.

Survey
The current survey was performed at 16 locations in Punjab, Pakistan. Bhalwal, Bumbi Zakhira Gashkori, Burj Jieway Khan, Chak 103 JB, Chak 22, Chak 35-2R/A, Chak Takht Hazara, Dera General Umro Khan, Islampur, Koat Maan Singh, Mazaharabad, Noorpur, Pull Bagar, Shamsabad, Sheikhupura, and 123 EB Pakpattan Canal were the locations surveyed ( Figure 1). A total of 210 soil and roots samples of maize and other rotational crops (potato, sorghum, wheat, tobacco, and green chilli) were obtained. The samples were collected during the spring season (March-April). Nematode distribution is rarely uniform or steady, and changes can happen quickly. The distribution of nematodes is usually uneven. Thus, the survey field that will be sampled was divided into subdivisions. Then, the effective sampling map was constructed. Large and small survey fields were sampled in a systematic, zigzag way. The main aim of the survey was to observe the prevalence and occurrence of nematode-related diseases.

Sampling
Root and soil samples were obtained to classify the distribution of nematodes while their higher populations were present [39]. Samples were collected at a depth of 10-15 cm with the aid of a hand shovel. Each sample contained 500 g of soil. The samples were stored in polythene bags and covered with rubber bands. All samples were appropriately

Sampling
Root and soil samples were obtained to classify the distribution of nematodes while their higher populations were present [39]. Samples were collected at a depth of 10-15 cm with the aid of a hand shovel. Each sample contained 500 g of soil. The samples were stored in polythene bags and covered with rubber bands. All samples were appropriately marked with a permanent marker on the location, such as date, host, locality, and any other related data, which later helped classify the samples' source.

Extraction of Cyst Nematodes and Eelworm Nematodes
The soil sample was approximately 500 g mixed, thoroughly stirred with water in a large bucket. Then, the suspension was left for 2-3 min to the deposition of soil particles. The suspension was passed through the 36 and then through 100 sieve mesh. The presence of cyst nematodes and several other long nematodes were deposited on these two sieves and were poured into a beaker. As described by Van Bezooijen [40], cyst extraction and counting were performed. For the extraction of these nematodes, the rest of the water contained an eel-shaped worm or nematodes; water passed through a 350-sieve mesh. Then, the suspension of 350 sieve mesh nematodes was spilled over a wet tissue paper mounted in a funnel so that water reached its bottom. The funnel was kept straight for 48 h to ensure the maximum population of nematodes. The 100 mL of nematode-containing water was drawn into a beaker after 48 h and analyzed under a stereoscopic binocular microscope [41].

Extraction of Root-Knot Nematodes
The roots were removed from the samples and sliced into 1-2 cm pieces. The roots were placed in a water-containing Petri dish. Every root piece was observed under the binocular microscope. Egg masses and females of root-knot nematodes (RKN) were removed by dissecting needles. Females were taken out cautiously, avoiding damage to their bodies. The collected females were moved to the cavity block with a dropper for further procedures, such as recognizing the species of Meloidogyne based on the perianal pattern. Root-knot nematode egg masses were transported to the cavity because larvae emerge from them, and their measurement helped to identify the species [40].

Identification of Nematodes
Standard morphological features were used in nematode identification. Different keys were used for the identification of nematodes at the species level [20,42]. For the higher categories and synonymy, Siddiqi's [43] book was followed for the order Tylenchida. Hunt's [44] classification was followed for the order Aphelenchida. The nomenclature provided by Jairajpuri and Ahmad [45] for the order Dorylaimida was used. Ahmad and Jairajpuri [46] nomenclature for the taxa of Mononchida was accepted for higher categories and synonyms in present details. However, Andrássy [47] and Andrássy's [48] classification was followed forRabditida. Moreover, to further confirm species, the life cycles of nematodes were observed in the greenhouse in pots.
2.6. Data Analysis 2.6.1. Community Analysis of Nematodes According to Norton [49], community analysis of nematodes was calculated from 16 localities in Punjab, Pakistan. Parameters such as Absolute frequency (AF%), Relative frequency (RF%), Relative density (RD%), and Prominence value (PV) were used to describe the nematode community structure. Cluster analysis of localities and nematodes, data based on the quantitative analysis (presence (1)/absence (0) of nematodes), which used to establish the similarity between localities based on Jaccard's coefficient of similarity. The dendrograms constructed based on data were related to the localities. All of the computations were conducted using the MINITAB (version 18) computer application. The diversity of nematodes was analyzed by one-way analysis of variance (ANOVA). The mean difference was calculated using Duncan's multiple range test (p > 0.05) for the significance test. All of the statistical processes were administered by different statistical packages such as IBM-SPSS statistics 25.0 version software and Microsoft Excel. Graphs were constructed through Sigma Plot 10.0 software.

Percentage Occurrence of Nematodes in Punjab, Pakistan
The overall proportion of plant-parasitic nematodes was 35% out of 210 soil samples of maize and other rotational crops, while free-living soil nematodes recovered 65% ( Figure 2). Locality-wise, the highest percentage of plant-parasitic nematodes in Bumbi Zakhira Gashkori was 70.65%, while the highest percentage of free-living soil nematodes in Pakistan was 95.03%. Moreover, percentage occurrence of Free-living soil and plantparasitic nematodes were 90% and 9.84% in Bhalwal; 29% and 70.65% in Bumbi Zakhira Gashkori; 33 Figure 3).

Statistical Analysis
The diversity of nematodes was analyzed by one-way analysis of variance VA). The mean difference was calculated using Duncan's multiple range test (p > the significance test. All of the statistical processes were administered by differen tical packages such as IBM-SPSS statistics 25.0 version software and Microso Graphs were constructed through Sigma Plot 10.0 software.

Percentage Occurrence of Nematodes in Punjab, Pakistan
The overall proportion of plant-parasitic nematodes was 35% out of 210 soil of maize and other rotational crops, while free-living soil nematodes recove ( Figure 2). Locality-wise, the highest percentage of plant-parasitic nematodes in Zakhira Gashkori was 70.65%, while the highest percentage of free-living soil ne in Pakistan was 95.03%. Moreover, percentage occurrence of Free-living s plant-parasitic nematodes were 90% and 9.84% in Bhalwal; 29% and 70.65% in Zakhira Gashkori; 33 Figure 3).

Occurrence (%) of Most Frequently Encountered Plant-Parasitic Nematodes from Different Localities
Soil samples of maize and other crops, those cultivated in sequence with the maize fields, showed that the three important genera of plant-parasitic nematodes, Heterodera Tylenchorhynchus and Helicotylenchus, were more prevalent and in a higher density than other plant-parasitic nematodes. Widespread occurrence (%) of plant-parasitic nema todes was followed, Heterodera zeae, with the highest occurrence (55.71%) followed by Tylenchorhynchus elegans (33.33%) and Helicotylenchus certus (24.76%). Heterodera zeae wa more prevalent than other plant-parasitic nematodes in maize plantations and othe crops cultivated in sequence with the maize fields ( Figure 4). It is a major pest of maize. I was found in the active form; it was observed in a condition when it was attached to the roots. However, their percentage occurrence varied at each surveyed site (Table 1).

Occurrence (%) of Most Frequently Encountered Plant-Parasitic Nematodes from Different Localities
Soil samples of maize and other crops, those cultivated in sequence with the maize fields, showed that the three important genera of plant-parasitic nematodes, Heterodera, Tylenchorhynchus and Helicotylenchus, were more prevalent and in a higher density than other plant-parasitic nematodes. Widespread occurrence (%) of plant-parasitic nematodes was followed, Heterodera zeae, with the highest occurrence (55.71%) followed by Tylenchorhynchus elegans (33.33%) and Helicotylenchus certus (24.76%). Heterodera zeae was more prevalent than other plant-parasitic nematodes in maize plantations and other crops cultivated in sequence with the maize fields ( Figure 4). It is a major pest of maize. It was found in the active form; it was observed in a condition when it was attached to the roots. However, their percentage occurrence varied at each surveyed site (Table 1).

Community Analysis of Plant-Parasitic and Free-Living Soil Nematodes
Community analysis of plant-parasitic nematodes and free-living soil nematodes showed high variability in population densities and frequencies of nematodes in different localities. However, during the studies, some nematodes were more frequently encountered in surveyed sites. Locality wise, some parameters of the community structure of free-living soil nematodes, and plant-parasitic nematodes of maize plantation and other crops, were cultivated in sequence with the maize fields at Punjab, Pakistan. These are given in Tables 2-17. Overall, the Heterodera zeae was a more frequently encountered species that is a major pest of maize. The nematode community structure of each locality contained parameters like; Absolute frequency (AF%), Relative Frequency (RF%), Relative density (RD%), and Prominence value (PV).

Community Analysis of Plant-Parasitic and Free-Living Soil Nematodes
Community analysis of plant-parasitic nematodes and free-living soil nematodes showed high variability in population densities and frequencies of nematodes in different localities. However, during the studies, some nematodes were more frequently encountered in surveyed sites. Locality wise, some parameters of the community structure of free-living soil nematodes, and plant-parasitic nematodes of maize plantation and other crops, were  Tables 2-17. Overall, the Heterodera zeae was a more frequently encountered species that is a major pest of maize. The nematode community structure of each locality contained parameters like; Absolute frequency (AF%), Relative Frequency (RF%), Relative density (RD%), and Prominence value (PV).                    The dendrogram generated due to 21 species of plant-parasitic and free-living nematodes depicts 7 cluster groups ( Figure 5). Nematode species found from localities 1, 5, and 15 (Bhalwal, Chak 22, and 123 EB Pakpattan Canal) constituted separate and independent groups, whereas few common species from locality 6 and 14 (Chak 35-2R/A and Sheikhupura), 7 and 8 (Dera General Umro Khan and Islampur) and 12, 11, 13 and 9 (Pull Bagar, Noorpur, Shamsabad, and Koat Maan Singh) cluster together formulating independent groups but with variable similarities and linkage properties. Common nematode species of localities 3 and 10 (Burj Jieway khan and Mazaharabad) also clustered with nematode species found from the other two localities viz., 2, and 4 (Bumbi Zakhira Gashkori and Chak 103 JB) but with the weakest possible linkage and similarities ties.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Potato
Six distinct groups can readily be recognized in the dendrogram ( Figure 6). Group 1, 4 and 5 developed as separate groups due to different nematode species recovered in 3 different localities viz., 3, 9 and 8 (Chak 103 JB, Pull Bagar and Noorpur, respectively). Group 2 comprises the common nematode species of three localities 2, 11 and 1 (Burj Jieway Khan, 123 EB Pakpattan Canal and Bumbi Zakhira Gashkori) with a comparatively higher similarities index compared to the other groups. Group 3 was formulated due to the highest similarities index among the common nematode species recovered from the 3 localities viz., 4, 5 and 7 (Chak 35-2R/A, Dera General Umro Khan and Mazaharabad). Group 6 comprises similar nematodes species found from the other two localities, viz., 10 and 6 (Sheikhupura and Islampur). The localities of group 2 have the Cephalobus nanus, Discolaimus texanus, Helicotylenchus certus and Heterodera zeae as the common nematode species, while Aphelenchus avenae, Cephalobus nanus, Discolaimus texanus and Rhabditis producta are similar nematodes isolated from three localities of group 3. Group 6 contained Aphelenchus avenae, Cephalobus nanus, Discolaimus texanus, Filenchus maqbooli n.sp., Heterodera zeae, Pratylenchus goodeyi and Rhabditis producta are common nematode species. H. zeae is present in 13 localities out of 15 localities. Heterodera zeae is the major pest of maize, and it was present in almost every field of maize, but its severity of infestation varies from locality to locality.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Potato
Six distinct groups can readily be recognized in the dendrogram ( Figure 6). Group 1, 4 and 5 developed as separate groups due to different nematode species recovered in 3 different localities viz., 3, 9 and 8 (Chak 103 JB, Pull Bagar and Noorpur, respectively). Group 2 comprises the common nematode species of three localities 2, 11 and 1 (Burj Jieway Khan, 123 EB Pakpattan Canal and Bumbi Zakhira Gashkori) with a comparatively higher similarities index compared to the other groups. Group 3 was formulated due to the highest similarities index among the common nematode species recovered from the 3 localities viz., 4, 5 and 7 (Chak 35-2R/A, Dera General Umro Khan and Mazaharabad). Group 6 comprises similar nematodes species found from the other two localities, viz., 10 and 6 (Sheikhupura and Islampur). The localities of group 2 have the Cephalobus nanus, Discolaimus texanus, Helicotylenchus certus and Heterodera zeae as the common nematode species, while Aphelenchus avenae, Cephalobus nanus, Discolaimus texanus and Rhabditis producta are similar nematodes isolated from three localities of group 3. Group 6 contained Aphelenchus avenae, Cephalobus nanus, Discolaimus texanus, Filenchus maqbooli n.sp., Heterodera zeae, Pratylenchus goodeyi and Rhabditis producta are common nematode species. H. zeae is present in 13 localities out of 15 localities. Heterodera zeae is the major pest of maize, and it was present in almost every field of maize, but its severity of infestation varies from locality to locality.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Po
Six distinct groups can readily be recognized in the dendrogram ( Figure 6). Grou 4 and 5 developed as separate groups due to different nematode species recovered different localities viz., 3, 9 and 8 (Chak 103 JB, Pull Bagar and Noorpur, respectiv Group 2 comprises the common nematode species of three localities 2, 11 and 1 ( Jieway Khan, 123 EB Pakpattan Canal and Bumbi Zakhira Gashkori) with a comp tively higher similarities index compared to the other groups. Group 3 was formul due to the highest similarities index among the common nematode species recove from the 3 localities viz., 4, 5 and 7 (Chak 35-2R/A, Dera General Umro Khan Mazaharabad). Group 6 comprises similar nematodes species found from the other localities, viz., 10 and 6 (Sheikhupura and Islampur). The localities of group 2 have Cephalobus nanus, Discolaimus texanus, Helicotylenchus certus and Heterodera zeae as common nematode species, while Aphelenchus avenae, Cephalobus nanus, Discolaimus anus and Rhabditis producta are similar nematodes isolated from three localities of gr 3. Group 6 contained Aphelenchus avenae, Cephalobus nanus, Discolaimus texanus, Filen maqbooli n.sp., Heterodera zeae, Pratylenchus goodeyi and Rhabditis producta are comm nematode species. H. zeae is present in 13 localities out of 15 localities. Heterodera ze the major pest of maize, and it was present in almost every field of maize, but its seve of infestation varies from locality to locality.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Sorghum
The dendrogram resulted from the clustering analysis of nematodes species of sorghum, in this case, given in Figure 7. Two main groups readily can be seen. Group 1 comprises the nematodes species isolated from soil samples collected from the rhizosphere around the roots of sorghum plants from two localities, viz., Burj Jieway Khan and Islampur (1 and 3). Group 2 was constructed due to the nematode species recovered from the other two localities of the same province, viz., Chak Takht Hazara and Sheikhupura (2 and 4). This group depicts its separate identity but comparatively lower linkage similarities among nematode species than group 1. It might be due to greater distance in the localities with variable climatic conditions and soil types. Common nematode species in groups 1 and 2 are Cephalobus nanus, Discolaimus texanus, Heterodera zeae and Tylenchorhynchus elegans. and Islampur (1 and 3). Group 2 was constructed due to the nematode species recovere from the other two localities of the same province, viz., Chak Takht Hazara an Sheikhupura (2 and 4). This group depicts its separate identity but comparatively low linkage similarities among nematode species than group 1. It might be due to great distance in the localities with variable climatic conditions and soil types. Common nem atode species in groups 1 and 2 are Cephalobus nanus, Discolaimus texanus, Heterodera ze and Tylenchorhynchus elegans.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Whe
The dendrogram developed from the data analysis showed two main clusters wheat ( Figure 8). Group 1 constituted due to nematodes species recovered from only th Burj Jieway Khan locality (1), while group 2 developed due to common nematodes sp cies encountered from the rest of the two localities, viz., Islampur and Sheikhupura and 3). The common species found in this group include Aphelenchus avenae, Cephalob nanus, Discolaimus texanus, Helicotylenchus certus and Rhabditis producta.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Wheat
The dendrogram developed from the data analysis showed two main clusters of wheat ( Figure 8). Group 1 constituted due to nematodes species recovered from only the Burj Jieway Khan locality (1), while group 2 developed due to common nematodes species encountered from the rest of the two localities, viz., Islampur and Sheikhupura (2 and 3). The common species found in this group include Aphelenchus avenae, Cephalobus nanus, Discolaimus texanus, Helicotylenchus certus and Rhabditis producta.
Sheikhupura (2 and 4). This group depicts its separate identity but comparatively low linkage similarities among nematode species than group 1. It might be due to great distance in the localities with variable climatic conditions and soil types. Common nem atode species in groups 1 and 2 are Cephalobus nanus, Discolaimus texanus, Heterodera ze and Tylenchorhynchus elegans.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Whe
The dendrogram developed from the data analysis showed two main clusters wheat ( Figure 8). Group 1 constituted due to nematodes species recovered from only t Burj Jieway Khan locality (1), while group 2 developed due to common nematodes sp cies encountered from the rest of the two localities, viz., Islampur and Sheikhupura and 3). The common species found in this group include Aphelenchus avenae, Cephalob nanus, Discolaimus texanus, Helicotylenchus certus and Rhabditis producta.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Tobacco
Analyzing different nematode species encountered from 3 localities generated 2 separate groups viz., 1 and 2 ( Figure 9). Group 1 includes common nematode species recovered from two localities, Chak 103 JB and Mazaharabad (1 and 3) on Euclidian distance basis. Group 2 comprises nematode species recovered from only one locality, viz., Chak Takht Hazara (2). Eleven plant-parasitic and soil nematodes were encountered from tobacco plants during the studies, while the Bitylenchus brevilineatus, Cephalobus nanus, Discolaimus texanus and Rhabditis producta are common nematode species which present in all localities. separate groups viz., 1 and 2 ( Figure 9). Group 1 includes common nematode species recovered from two localities, Chak 103 JB and Mazaharabad (1 and 3) on Euclidian distance basis. Group 2 comprises nematode species recovered from only one locality, viz., Chak Takht Hazara (2). Eleven plant-parasitic and soil nematodes were encountered from tobacco plants during the studies, while the Bitylenchus brevilineatus, Cephalobus nanus, Discolaimus texanus and Rhabditis producta are common nematode species which present in all localities.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Green Chilli
A total of 10 species recovered from the soil around the roots of chilies plants from 3 different localities, viz., Chak 22, Chak Takht Hazara and Islampur (1, 2 and 3), were found clustered into the three distinct groups ( Figure 10). Hence, all of the resultant groups contain different nematode species. The dissimilarities among nematodes might be because of different cropping patterns followed, water qualities, soil structure, and texture.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Green Chilli
A total of 10 species recovered from the soil around the roots of chilies plants from 3 different localities, viz., Chak 22, Chak Takht Hazara and Islampur (1, 2 and 3), were found clustered into the three distinct groups ( Figure 10). Hence, all of the resultant groups contain different nematode species. The dissimilarities among nematodes might be because of different cropping patterns followed, water qualities, soil structure, and texture. recovered from two localities, Chak 103 JB and Mazaharabad (1 and 3) on Euclidian distance basis. Group 2 comprises nematode species recovered from only one locality, viz., Chak Takht Hazara (2). Eleven plant-parasitic and soil nematodes were encountered from tobacco plants during the studies, while the Bitylenchus brevilineatus, Cephalobus nanus, Discolaimus texanus and Rhabditis producta are common nematode species which present in all localities.

Cluster Analysis of Localities Regarding Nematodes Species Encounter from Green Chilli
A total of 10 species recovered from the soil around the roots of chilies plants from 3 different localities, viz., Chak 22, Chak Takht Hazara and Islampur (1, 2 and 3), were found clustered into the three distinct groups ( Figure 10). Hence, all of the resultant groups contain different nematode species. The dissimilarities among nematodes might be because of different cropping patterns followed, water qualities, soil structure, and texture.

Discussion
Nematodes co-exist in different ecosystems, but their occurrence, density, and distribution depend largely on environmental and edaphic variables [50,51]. Nematodes can cause severe damage to maize. For community indicator analysis, nematodes can form the most relevant group because of more data about their taxonomy and feeding categories than other macro-fauna. Free-living nematodes are very effective and necessary for degrading organic materials and recycling soil nutrients. Free-living nematodes are essential for plant growth [52]. However, plant-parasitic nematodes inflict massive losses to the wide range of crops due to their yield and growth [53]. They can destroy crops through nurturing and by connecting with other microbes, which presents a major threat to crops, with an expected annual loss of USD 100-150 billion [54]. More than 120 species of nematode parasitize maize, and some of these species are known as primary pathogens [55][56][57]. Several major genera of plant-parasitic nematodes have been reported associated with maize: Criconemella, Ditylenchus, Globodera, Haplolaimus, Helicotylenchus, Hemicriconemoides, Heterodera, Longidorus, Meloidogyne, Paratrichodorus, Pratylenchus, Radophulus, Rotylenchulus, Scutellonema, Trichodorus, Tylenchorhynchus and Xiphinema [58].
However, the present findings confirm the results of previous surveys carried out in different countries by many researchers that worked on similar objectives viz., [59,60]. The present study revealed that 24 species were identified from maize and other rotational crops from 16 localities through Punjab, Pakistan. During the study, the most abundant plantparasitic nematodes were, Heterodera zeae, with the highest incidence (55.71%), followed by Tylenchorhynchus elegans (33.33%) and Helicotylenchus certus (24.76%). However, the present findings confirm the results of previous surveys carried out by Mirsam, Muis and Nonci [60] that the Heterodera spp., Tylenchorhynchus spp. and Helicotylenchus spp. showed comparatively high frequency and density than for other trophic groups of nematodes in maize. Many researchers described that the cyst-infected maize exhibits extensively poor growth, is easily pulled out from the soil, is usually about half the size of healthy plants, forms patches in the field, discolors leaves turning yellow and sometimes turning reddish from tips that yield less grain than healthy plants [61][62][63][64][65]. During the present study, H. zeae was more prevalent in the maize field and other crops cultivated in nearby maize fields. Maize and other crops from nearby maize areas were invaded by several major nematodes that may cause numerous diseases.
During this study, the other rotational crops (such as potato, sorghum, wheat, tobacco and green chilli) were also studied because these crops were cultivated in sequence with the maize in the same field at the time of the survey. These crops have economic importance, but unfortunately, they suffer from several plant-parasitic nematodes. The previous studies reported that the cyst nematodes (Heterodera spp.), root-knot nematode (Meloidogyne spp.), stunt nematodes (Tylenchorhynchus and Bitylenchus spp.), spiral nematodes (Tylenchorhynchus spp.), lesion nematodes (Pratylenchus spp.), reniform nematodes (Rotylenchulus spp.), sheath nematodes (Hemicriconemoides spp.), rot nematode (Ditylenchus spp.), sting nematode (Belonolaimus spp.), stubby-root nematode (Trichodorus spp.), lance nematodes (Hoplolaimus spp.), foliar nematodes (Aphelenchus and Aphelenchoides spp.), ring nematodes (Criconemoides spp.) and dagger nematodes (Xiphinema spp.) are major plant-parasitic nematodes which are associated with potato, sorghum, wheat, tobacco and green chilli [22,[66][67][68][69][70][71]. Moreover, during the present study, all of these nematodes were frequently present in all rotational crops. Migratory endoparasitic nematode, Pratylenchus reduced chlorophyll content, plant height, shoot and root weight also the destruction of the cortical parenchyma and epidermal tissues leading to severe root necrosis in maize [24,72,73]. During the present study, the Pratylenchus sp. was observed in maize and the other rotational crops of maize. Elhady, et al. [74] reported that the Pratylenchus penetrans was an endoparasite that invaded and migrated through roots as a juvenile or adult without becoming sedentary and escaped to the soil when conditions inside roots become adverse. Our previous study reported one new species Filenchus maqbooli, five new record species Helicotylenchus certus, Helicotylenchus gulabi, Helicotylenchus jasminii, Pratylenchus goodeyi and Telotylenchus indicus and two new host record Tylenchorhynchus tritici and Malenchus labiatus from maize [75].
The reniform nematode Rotylenchulus species commonly causes damage in tropical environments, and it causes 40-60% yield losses [24]. Helicotylenchus and Tylenchus were observed in maize and bean cropping systems [76]. However, Tylenchus spp. is not considered a major limiting factor in maize production even at high populations, probably because their economic importance has not been entirely ascertained [77]. Moreover, Helicotylenchus was the most frequently occurring genus in maize due to its cosmopolitan and polyphagous nature [78]. The high density of Helicotylenchus was correlated with reducing chlorophyll content, root mass and plant height in maize [79]. The migratory ectoparasites, namely, Longidorus and Xiphinema occurred in low abundance in this study. Our findings are consistent with Atandi, Haukeland, Kariuki, Coyne, Karanja, Musyoka, Fiaboe, Bautze and Adamtey [76], who found a low abundance of Longidorus and Xiphinema in maize and beans fields in Kenya. Our results correspond with Mokbel [80] recorded the following species associated with maize, namely Criconemella sp., and Helicotylenchus sp., Heterodera sp. grown in Abu-Arish governorate, Jizan province, Southwest, Saudi Arabia. Cluster analysis was also performed based on nematode communities in maize and other rotational crops. The resulting dendrograms showed a diversity level among the different localities of the maize and other rotational crops regarding the presence of nematodes species.

Conclusions
The present study reveals that 24 species of nematodes are present in maize and other rotational crops. Most of the species reported in this study are highly pathogenic, and thus their occurrence can pose severe threats to maize and other rotational crops and require the urgent attention of researchers. Special attention must be paid to the nematode attack hotspots found in the present case. The above will need to keep in mind the susceptibility level of these crops to the PPNs existing in the soils and ecosystems in which the plantations will need to be established. The outcomes of this research could be valuable in the future for identifying potential perturbations in the maize, such as agronomic methods, the application of biological control agents. In the future, this study will be a base for more research into the estimation of crop yield losses in maize and other crops cultivated with maize. This study underlined plant-parasitic and free-living nematodes variability and community structures as essential indicators for evaluating resilient approaches in maize growing systems. Further investigations can also concentrate on community rearrangements and relationships within species co-existence processes to establish methods for protecting or preserving diversity rather than removing the most pathogenic species.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article.