Plant Parasitic Nematodes Associated with Three African Indigenous Vegetables in Southwest Nigeria

Background and Objective: African indigenous vegetables are rich in nutrients and medicinal properties that are important for health and vitality, but their availability is on the decline partly due to attacks by plant parasitic nematodes, hence field surveys were conducted in 2016 and 2017 to investigate the distribution of plant parasitic nematodes and their interactions with free-living nematodes in fields planted to three African indigenous vegetables in Southwest Nigeria. Materials and Methods: A total of 180 soil samples were taken from 180 farms in all Local Government Areas (LGAs) visited in the four States in Southwest Nigeria. Samples were taken from three vegetable fields in all the LGAs visited. Nematodes were extracted from 200 mL sub-samples; the nematodes were counted and identified under a compound microscope using a pictorial guide. Results: The results showed that 16 genera of plant parasitic nematodes were found associated with Amaranthus cruentus, Solanum macrocarpon and Telfairia occidentalis . Meloidogyne , Helicotylenchus , Rotylenchulus , Xiphinema , Pratylenchus and Hoplolaimus were the most abundant nematode species encountered in vegetable fields in the study areas. The correlation analysis shows an antagonistic relationship between free-living nematodes and plant parasitic nematodes. Conclusion: There is a need to cultivate vegetable crops to suppress the populations of plant parasitic nematodes in Southwest Nigeria.


INTRODUCTION
Telfairia occidentalis, Solanum macrocarpon and Amaranthus cruentus are among the premium African indigenous vegetables grown in Southwest Nigeria.Their presence in nearly every market in this region describes how useful and important they are in the diet of the people.They are known to contain important minerals, vitamins, proteins and medicinal constituents that are relevant to human growth and well-being 1,2 .Amaranthus cruentus is rich in vitamin A and important for vision, particularly in children 1 .Solanum macrocarpon is known to be bitter and used as worm expellant for the treatment of respiratory and cardiovascular diseases 3,4 .
Telfairia occidentalis is rich in iron, hence it is in high demand by lactating mothers and anemic patients 5 .
Generally, these vegetables are leafy vegetables and as such are used in making soups and stews that can be eaten with various staples, thereby, providing essential balance diets to many Sub-Saharan Africans, who cannot afford meat, milk, egg and other proteins of animal origin 6,7 .It is because of the nutritional and medical importance of these vegetables that World Health Organization recommends that their consumption rate should be about 400 g per person per 8 .They are also important source of income, particularly to rural poor farmers 9,10 .
However, their production is limited by both marginal soils and diseases.A number of nematodes have been reported on many vegetables [11][12][13][14][15] .Adekunle et al. 14 reported a significant reduction in biomass yields of T. occidentalis following attack by Meloidogyne incognita, Tylenchus spp., Helicotylenchus spp., Hirschmaniella spp.and Longidorus spp., while Ogundele et al. 15 reported the reduction of leaf yields of A. cruentus and T. occidentalis by Meloidogyne incognita, Helicotylenchus spp.and Dolichodorus spp.
Many nematodes are soil-borne pathogens, they infect plants from the roots and through their feeding habits, they deform the roots of plants, hence interfering with the normal functions of the root system.
They can also form disease complexes with other pathogens thereby increasing the severity of disease and making control difficult 16,17 .
The populations of plant parasitic nematodes can be affected by the availability of free-living soil microbes including free-living nematodes and opportunist microorganisms 18,19 .Khan and Kim 18 reported a decrease in the populations of M. incognita following an increase in the populations of Mononchoides striatus, however; this relationship depends on soil conditions.More so, Amulu et al. 19 reported that they were high populations of free-living nematodes in comparison with the populations of plant parasitic nematodes in fadama fields rich in organic matter.They proposed that the activities of the free-living nematodes might have contributed to the low populations of plant parasitic nematodes.This study investigated the distributions of plant parasitic nematodes and their relationship with free living nematodes associated with three African indigenous vegetables grown in organic soils in Southwest Nigeria.LGAs) and two in Ondo (Akoko Northeast and Akoko Southwest LGAs).A total of fifteen LGAs were visited for soil sampling.In each LGA, samples were taken from four farms.Sixty soil samples were taken in fields sown to A. cruentus, S. macrocarpon and T. occidentalis giving a total of 180 samples in all the States visited.

Field
The number of plots in a farm ranged from two to six plots of 17×11 m each per farmer.The vegetables were cultivated on 2×3 m beds.The farmers were provided with pelletized organic fertilizer (sunshine, manufactured by Ondo State Government).The organic fertilizer was applied to all plots in each farm at the same rate (10 ton/ha) by all the farmers in each LGA.Soil samples were taken six weeks after planting.Soil samples were collected from the root zones of 20 vegetable crops using zigzag sampling method in all the farms hat were visited in the study area.This was done using a soil auger that has a depth and diameter of 15 and 1.9 cm respectively.Soil samples collected in each farm were added together to form a composite sample and 200 mL sub-sample was taken from the composite sample for nematode analysis.Nematodes were extracted from the 200 mL sub-sample using the modified Baermann tray method 20 .Recovered nematodes from the soil samples in water suspension were killed by adding equal quantity of boiling water into the nematode suspension and fixed in 4% (w/v) formaldehyde 21 .Nematodes were placed in a counting dish and counted under a stereomicroscope (100×) (Motic SMZ-160M Motic-Group, China).After these nematodes were identified individually from each sample under a compound microscope (100×) (Motic microscopes; model: 61400131, Motic-Group, China) to genus level, with the aid of identification manual to genera of nematodes 20,22,23 .The sampling was repeated in 2017, during the same months and at the same farms, as the first year sampling was carried out.Percentage frequency of nematode distribution and percentage nematode population density were calculated using the following formula: Where, n is the number of times an individual nematode occurred in all the samples and N is the sample size: In Nematode population = ×100 TN Where, In is the individual nematode population in all the samples and TN is the total population of all the nematodes extracted from all the samples.Nematode population counts were log-transformed [Log10 (x+1)] and subjected to Pearson's correlation analysis to establish relationships among nematode species using the Statistical Analysis System (SAS) package 24 .Nematode genera were classified as outlined by Hunt et al. 25 .---------------------------------------------------

DISCUSSION
The results of the current study show that sixteen genera of plant parasitic nematodes were isolated and identified in fields grown to A. cruentus, S. macrocarpon and T. occidentalis in Southwest Nigeria.Meloidogyne spp., Helicotylenchus spp., Rotylenchulus spp., Xiphinema spp., Hoplolaimus spp.and Pratylenchus spp.were the most prevalent nematode species encountered in the study area.The results of these findings agreed with the findings of Atungwu et al. 13  Large numbers of these plant parasitic nematodes were extracted from soil samples taken around the roots of the vegetables across the four States.These nematodes have consistently been encountered in a number of vegetable fields [27][28][29] .The root-knot nematode, Meloidogyne spp.had been reported to parasitize and cause significant yield losses on a number of vegetables globally 30,31 .Meloidogyne incognita is the most common root-knot nematode species in Nigeria and the most damaging on a worldwide basis 32 .
About 80% yield losses due to this nematode on vegetables had been reported in heavily infested soils 33 .Severe damage to vegetables by the root knot nematode, M. incognita has been reported in many African countries 34 .Significant yield reductions of okra by the nematode have been reported in Nigeria 35 .
A recent study in Southwest, Nigeria showed that the interactions between Helicotylenchus and other plant parasitic nematodes resulted in `significant yield losses in some vegetable fields 14,15 .The nematode is known to prefer soils rich in organic matter 36 .This is probably because their proliferation and reproduction are enhanced by nitrogen 37 .This might have contributed to the high populations recorded in various communities, where samples were taken as organic fertilizer (sunshine) rich in nitrogen was used by the farmers.
Rotylenchulus spp. was encountered in large numbers in many of the communities where these vegetables were grown.It is known that after Meloidogyne spp., R. reniformis is the most economically important nematode affecting vegetable production in the tropics 38 .This may be attributable to the fact that the nematode also induces gall-like swellings known as syncytia on root systems like Meloidogyne spp.; hence many workers and farmers might have misdiagnosed the infections caused by Meloidogyne spp.In the present study, the nematode was among the most widely occurring and abundant nematode species encountered in the study areas.In Nigeria, the nematode is not recognised as important pest of vegetables 38 .The nematode has been found parasitizing Solanum spp., Amaranthus spp.amongst other indigenous vegetables in Nigeria and elsewhere [30][31][32] .
Pratylenchus spp. was also found across the states in A. cruentus, S. macrocarpon and T. occidentalis fields.
The most reported Pratylenchus spp. in Nigeria is P. brachyurus and it has been found in large numbers in vegetable fields 38 .Damage caused by Pratylenchus spp.has been documented in a number of vegetable crops including tomato, eggplant and cucumber in the temperate regions 23 .However, the nematode is not recognised as an important pest of vegetables in Nigeria due to the overriding importance of root-knot nematode in vegetable fields 38 .In several studies conducted in many African countries, Pratylenchus spp.were found to be associated with a number of vegetables and in some cases it was recorded as a major pest in the study areas [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] .Xiphinema, Longidorus and Paratrichodorus sp. were the most important ectoparasitic nematodes reported to be associated with the three vegetables in some of the States sampled.Xiphinema spp.were the most abundant and the most widely distributed in the study areas when compared with Paratrichodorus spp.Soil texture is one of the main edaphic factors affecting the distribution of these nematodes.They are known to be more active and reproduce effectively in lighter soils 39 .The highest population densities of these pests were recorded in derived savannah areas of the region.This area is characterised by light soil and fairly low rainfall and this might have favoured their activities and reproduction.Apart from their direct effects on crop plants, they can also transmit viruses through their feeding habits thereby, exacerbating disease infections 28,40 .They are however, not recognised as important pests of agricultural crops in Nigeria 38 .These nematodes have been reported associated with a number of vegetable crops including tomato, cucumber, aubergine, S. melongena and sweet pepper, green beans and squash 41 .
Dolichodorus spp. was encountered in communities in Ondo State.The nematodes are not common in vegetable fields in Nigeria and as such little or no information is known with reference to their effects on vegetables in Nigeria.However, the nematode can cause damage as severe as those caused by the sting nematode Belonolaimus longicaudatus, however, their activities are limited to moist habitats, hence yield losses caused by the nematode is not widely spread 42 .In this study the nematodes were encountered in hydromorphic fields.The nematode has been shown to reduce the yields of T. occidentalis and A. cruentus in Nigeria 15 .
Scutellonema species were encountered in low populations and were among the least distributed and least abundant.The nematodes were however, encountered only in A. cruentus fields.This pest is not known to cause damage in vegetable fields in the tropics [43][44] but a number of authors have reported the devastating effects of Scutellonema Brady in tuber crops particularly yam tubers 43,44 .The presence of Scutellonema spp. in vegetable fields could be due to farming practices and cropping history as some of the farmers practice mixed cropping, where they combined these vegetables with other crops.However, Scutellonema clathricaudatum was reported as third most prevalent nematode affecting vegetable crops in Benin 27 .The authors reported that the nematode infected 53.9% of the crops sampled and was observed on vegetable crops in 44.4% of surveyed communities.
Other nematodes that were encountered in the study areas were Hoplolaimus, Criconemoides, Hemicriconemoides, Tylenchus, Tylenchorhynchus, Aphelenchus and Hemicycliophora species.Though they are not considered as important nematode pests affecting vegetables in Nigeria, but have been reported on a number of vegetables in both the tropical and subtropical regions of the world including Nigeria 27,41,45 .
The frequency of occurrence of these nematodes on crops including vegetables differs.This may be attributable to a number of factors including root exudates produced by some of these vegetables and soil conditions.Take for instance Xiphinema was not reported in soil samples taken from around the roots of T. occidentalis but were reported in those taken from around the roots of A. cruentus and S. macrocarpon in Ekiti State.It is possible that the nematode does not prefer feeding on this vegetable due to its antagonistic nature.Authors have demonstrated that some plant roots produce substances that antagonistic to the nematode.
The numbers of nematodes in this current study are generally low; this may be due to the fact that soil sampling periods were during the growth period of the investigated crops and some of the nematode species are endoparasitic in their feeding habit hence method of extraction may not display the whole plant parasitic community.
The correlation analysis results in the present study further confirm the antagonistic relationship between free living and plant parasitic nematodes.Fields sown to the three vegetables in the three States revealed The applications of the organic fertilizers by the farmers might have influenced this interaction.
This finding agrees with those of Moosavi and Zare 46 , who reported significant increase in the populations of bacteria feeding nematodes, fungi feeding nematodes, omnivorous feeders and predatory nematodes with corresponding decrease in the populations of plant parasitic nematodes following the application of sun hemp as green manure in plots sown to cucumber.Our earlier study also showed that the incorporations of sun hemp and Mexican sunflower seedlings at 10 or 20 seedlings/plot each significant increase the population of Rhabditis, Mononchus and Dorylaimus spp. in comparison with fallow control plots sown to S. macrocarpon, A. cruentus and T. occidentalis in soils amended with sun hemp and Mexican sunflower 47 .The populations of the free living nematodes might have been stimulated by the organic fertilizers used by the farmers.Authors have demonstrated that the applications of organic materials in soils can enhance the build-up of free living nematodes; hence, it is possible that the organic fertilizer enhanced the populations and activities of the free living nematodes in this study, which in turn affected the populations of the plant parasitic nematodes 48 .
Mononchus spp., may affect the nematode population directly by feeding on the plant-parasitic nematodes, but Rhabditis (bacteria feeders) and Dorylaimus (omnivorous feeders) spp.may not directly affect the populations of plant parasitic nematodes but their feeding activities may result in the release of natural chemical substances that may be lethal to plant parasitic nematodes.Dorylaimus is known to play important role in the degradation of organic matters and during this process a number of toxic metabolites such as ammonia, flavonoids, nimbin, azadirachtin and phenols are released into the soil and these in turn create unfavorable conditions for plant-parasitic nematodes 48 .The predatory nematode, Mononchus spp.can affect nematode populations directly; it is known to possess massive teeth-like structure known as onchia which they use in killing plant parasitic nematodes.
In the correlation analysis, there are some positive correlations between plant parasitic nematodes such as Hoplolaimus and Meloidogyne (0.939), Rotylenchulus and Hoplolaimus (0.939) this is an indication that the activities of one plant-parasitic nematode may also increase the populations of another plant-parasitic nematode.
Continuous cultivation of vegetables in a particular field could trigger and facilitate the buildup of plant parasitic nematodes and this might consequently affect crop yields.The cultivation of vegetables could be beneficial in organic soils, fadama fields or soils amended with organic materials.The reason being that, these soils favor the multiplication of free living nematodes and other microorganisms that are antagonistic to plant parasitic nematodes and this will in turn keep their population below economic thresholds.

CONCLUSION
Among the sixteen genera of plant parasitic nematodes found associated with the vegetables, Meloidogyne, Helicotylenchus and Rotylenchulus spp.Pratylenchus spp.and Xiphinema spp.were the most prominent nematode pests in the region.Free-living nematodes significantly correlated negatively with plant parasitic nematodes.Farmers are therefore advised to cultivate their vegetables in ways and manner that will not support the build-up of nematode pests beyond economic thresholds.One way of achieving this is through the use of organic amendments in growing their crops as this can act as both fertilizers and natural nematicides and can also facilitate the build-up of free-living nematodes.Further study should be conducted to measure the damage and yield losses these nematode pests can cause on the three African indigenous vegetables.

SIGNIFICANCE STATEMENT
This study investigated the distributions of plant parasitic nematodes and their relationship with free living nematodes associated with three African indigenous vegetables grown in organic soils in Southwest Nigeria.The result revealed that 16 genera of plant parasitic nematodes were found associated with three African indigenous vegetables in Southwest Nigeria.The correlation analysis shows an antagonistic relationship between free living nematodes and plant parasitic nematodes.

Plant parasitic nematodes belonging to three orders Rhabditida, Dorylaimida and Triplonchida, 3 sub-orders Tylenchina, Dorylaimina and Diphtherophorina, four sub-families Aphelenchoiddoidea, Tylenchoidea, Dorylaimoidea and Diphtherophoroidea and ten families Aphelenchidae, Tylenchidae, Dolichodoridae, Hoplolaimidae, Meloidogynidae, Pratylenchidae, Criconematidae
, Hemicycliophoridae, Longidoridae and Trichodoridae were encountered and identified in Southwest Nigeria.Sixteen genera of plant parasitic nematodes were encountered in soil samples taken from around the root zones of the three African indigenous vegetables in Southwest Nigeria in 2016.Helicotylenchus spp.and Pratylenchus spp.were the most abundant nematode species in soil samples taken from around the root zones of the vegetable with 1574 and 1421/200 mL soil respectively in Ekiti State.Meloidogyne spp.had high frequency rating of 83% but low population density of 313/200 mL soil.The populations of other nematode species including Hoplolaimus spp., Aphelenchus spp., Longidorus spp., Hemicycliophora spp., Criconemoides spp.and Hemicriconemoides encountered in this State (Ekiti) were low ranging from 11-141/200 mL soil.The populations of Hoplolaimus spp.were however, high in S. macrocarpon fields in 2016 and 2017.

Table 1 :
Plant parasitic nematode genera associated with three indigenous vegetables in Ekiti State

Table 4 :
28equency of occurrence, Figures in parenthesis are means of percentage nematode populations and sample size28and Hoplolaimus and negatively with Mononchus.Mononchus correlated negatively with Meloidogyne.The results of the 2017 followed similar trend with those of 2016, except that Pratylenchus correlated positively with Rhabditis, Dorylaimus and Mononchus and negatively with Hoplolaimus (Table6).Rhabditis correlated positively with Mononchus, Dorylaimus and negatively with Hoplolaimus.Dorylaimus correlated positively with Helicotylenchus and Mononchus.Plant parasitic nematode genera associated with three indigenous vegetables in Oyo State 2016 2017 - In Telfairia occidentalis field: In 2016, Helicotylenchus correlated negatively with Meloidogyne and positively with Rhabditis, Mononchus and Dorylaimus (Table 5).Rhabditis correlated negatively with eloidogyne and positively with Mononchus and Dorylaimus.Mononchus correlated negatively with https://doi.org/10.17311/tas.2024.69.82 | Page 74 28equency of occurrence, Figures in parenthesis are means of percentage nematode populations and sample size28

Table 6 :
Correlation analysis between plant parasitic and free-living nematodes in fields grown to three indigenous vegetables in Southwest *Significant at 5% level of probability,**Significant at 1% level of probability, -: Negative correlation and +: Positive correlation 15milarly, Adekunle et al.14and Ogundele et al.15identified the presence of 3 genera of plant parasitic nematodes including M. incognita, Dolichodorus, Longidorus, Helicotylenchus and Xiphinema species each affecting T. occidentalis and A. cruentus in vegetable fields in Osun State Nigeria.The former reported significant reduction in biomass of T. occidentalis planted in fields infested with M. incognita, Longidorus and Xiphinema species while the latter reported significant reduction in leaf yields of A. cruentus and T. occidentalis grown in fields infested with M. incognita, Helicotylenchus and Dolichodorus spp.In Ebonyi State, Southeast Nigeria, Ngele and Kalu 26 revealed the presence of seven genera of nematodes including M. incognita (35.5%),Pratylenchus spp.(20.83%),Heterodera spp.(15%), , who revealed the presence of five nematode genera in fields usually planted to three leafy indigenous vegetables in Ogun State Nigeria.The identified nematodes were; Tylenchus, Pratylenchus, Helicotylenchus, Meloidogyne and Rotylenchulus spp. on Celosia, https://doi.org/10.17311/tas.2024.69.82 | Page 76 Amaranthus and Corchorus.
Mononchus and Dorylaimus spp.population densities correlated negatively with those of some genera of plant-parasitic nematodes including Meloidogyne, Hoplolaimus and Rotylenchulus spp.