PLANT PARASITIC NEMATODE OF GENERA APHELENCHUS AND APHELENCHOIDES (NEMATODA: APHELENCHOIDEA) FROM DISTRICT LAKKI MARWAT, KHYBER PAKHTUNKHWA, PAKISTAN Samreen Khan, Salma Javed, Tabassum Ara Khanum National Nematological Research Centre, 9University of Karachi, Karachi-75270, Pakistan. Corresponding author E- mail: sajaved @uok.edu.pk ABSTRACT The research work is based on the study regarding nematodes belonging to the order of Aphelenchida. Research work was carried out in four sites of District Lakki Marwat, Khyber Pakhtunkhwa Pakistan, where no such research was conducted about nematode fauna presence in the past. For the said purpose, numbers of soil samples from different rhizosphere were collected from various sites in the year 2019. Upon completion of all pre-requisit, the related correspondence and data of the surveyed sites with their results including, occurrence percentage, Relative Frequency, Simpson Diversity Index and Dendrogram of Cluster Analysis were ensured. However, detailed analysis revealed two types of genera i.e Aphelenchus (49%) while Aphelenchoides (51%). Resultantly, two species of Aphelenchus namely Aphelenchus avenae and Aphelenchus isomerus; while four species of Aphelenchoides namely Aphelenchoides bicaudatus, Aphelenchoides richardsoni, Aphelenchoides unisexus and Aphelenchoides macrospica were surfaced. The Aphelenchus avenae was found most frequently abundance species (40%) from all sites and followed by Aphelenchoides richardsoni (20%). The highest abundance occurrence site of both genera was village Aba Khel (50% each) and the lowest one village Aghzar Khel Aphelenchus (22.7%) and Aphelenchoides (18.1%). All these species were previously reported for Pakistani nematode fauna except Aphelenchoides macrospica which was surfaced for the frist time from Pakistan. It is also pertinent to mentioned here that remaining species were surfaced from new host and location from Pakistan. Key words: Aphelenchus, Aphelenchoides, District Lakki Marwat, New record, New host and locality. INTRODUCTION 2Lakki Marwat is one of the Southern District of Khyber Pakhtunkhwa Province, Pakistan and located at 32°17’- 32°53’ N Latitude and 70°23’ - 171°16’ E Longitude. Total area covered of 3,164 Squre kilometers (ESA, 2012) and having population of 876,182 as per last census conducted in year 2017. 1On the North side covered by District Bannu and District Karak while on the East side bounded by District Mianwali, Panjab. Towards South East of the District Dera Ismail Khan while towards Southwest covered by the District Tank. On the Western side joined by the tribal areas of District Tank including some area of South Waziristan Agency. Region of district Lakki Marwat 2has all the characteristics of a desert due to its sand dunes, scorching heat and dry weather with hot summers and moderately cool winters. However, 2rainfall is very rare and intermittent and generally arise in July and August. The component elements of the area’s soils are extremely and deeply drained, calcareous and uneven surfaced and likewise loamy sand. 1The fine textured soils are sandy clay and clay loam of the area and contained variety of rocks of Bhittani and Waziristan ranges. Climate of the12area is arid to semi arid and sub-tropical continental. Variation in temperature scale commonly noted during the summer season from 27-42ºC and in winter season from 4-20ºC (Haq, 2009). Geography 2is a mixture of hills and sandy plains. Maximum boundries of the district contained on hills including East, Southeast, Southwest as well as Northwest. General dimension and 1elevation of these hills ranging from 500 to 1000 meters and most of the region is almost shown 200 to 300 meters above level of sea. Most of region consists on arid plains and required a lot number of sources which is appropriate and commonly considered fruitfull for the development of agriculture field. However, the area unfortunately facing acute shortage in constraints i.e shortage of water and non existance of irrigation system due to which the land is extremely weak for agriculture purpose (ESA, 2012). Lakki Marwat area is not rich with any sources to highlight but entirely depends upon rain water. The two prominent crops are wheat and gram and mostly the drought has severely affected both 5crops in the sandy areas of district. These crops have been grown on vast tract of land in the barani (rain fed) belt. The crops especially wheat and gram have been affected due to low perspiration in winter season, which is a major cause of concern for farmers. Other than aforementioned; some other crops are also growing up including maiz, rice, Bajra, Jowar (Sorghum), Sugarcane, Gaura, Barley, Rape and Mustared, Masoor, Dates, Orange, Mango, Gauva, Watermelon, Melon, Pomegranates, Banana, Grapes, Mulberry and Ber. The planet earth is engrossed by immense diversity of life and existence of nematodes are the second most species rich phylum next to Arthropoda (Keshari et al., 2019). Nematodes are parasites and microscopic round worms and it may be source of threat to food insecurity in numbers of countries. Nevertheless, Pakistan face the issues of damaging of plants by nematodes due to its geographical location particularly in the sultry areas in which the climate is considered utmost appropriate for reproduction including activities of nematodes all over the year (Maqbool, 1988). The Order Aphelenchida is one of the abstemiously limitless nematodes order which is consists on numbers of ecological sets including fungal feeding, obligate insects’ parasites, predator while some of them belongs to phytoparasite related to 3the genera Bursaphelenchus Fuchs, 1937 and Aphelenchoides Fischer, 1894 (Nickle, 1970), but large number of species of Aphelenchoides15are fungal feeder or predators (Kanzaki and Giblin-Davis, 2012). Generally, the aphelenchs are highly active nematode, predominantly mycetophagous in habit, cosmopolitan in soil, plant debris free-living and association with lichens, on tree bark as well as in the tunnels of wood-boring beetles (Hunt, 1993). Aphelenchs belong to be genera Aphelenchus and Aphelenchoides are the utmost and widely spread and being primarily mycetophagous almost certainly represent the nearby to the ancestral forms (Hunt, 1993). Although, most Aphelenchoides are entirely mycetophagous, while few species under the sobriquet ‘bud and leaf nematodes’, parasitize higher plants. However, Aphelenchoides may be retrieved from mosses, soil, decaying organic materials, mushrooms while from plant tissues (Khusainov, 2013). 14Sanchez- Monge et al., 2015 search out plant parasitic nematodes with presented 13 plant parasites considered most important all over the world. Currently, nineteen species of genus Aphelenchoides Fischer, 1894 and four species of genus Aphelenchus Bastian, 1865 has been reported from Pakistan (Shahina et al., 2019). The objective of this plan was to study the Aphelenchid nematodes by conducted nematological survey from different areas of District Lakki Marwat, Khyber Pakhtunkhwa (KPK), Pakistan. MATERIALS AND METHODS Area of Exploration: Nematological survey was undertaken in the months of October and November 2019 in various sites including villages Aba Khel, Aghzar Khel, Sarai Naurang and Tajori of the district Lakki Marwat, Khyber Pakhtunkhwa, Pakistan. Total 61 soil samples of different plants were retrieved and subsequently these samples were safely brought to 13laboratory of National Nematological Research Centre, University of Karachi, Pakistan. The aim of said survey was to establish the biodiversity of Aphelenchid nematodes. Collection and Processing of Soil Sample: The soil samples (500 gm) each from different infected plant host were collected and packed in clean plastic bags, tied up and labeled appropriately containing relevant information i.e. host, locality, time and date of collection and shifted to the laboratory of 4National Nematological Research Centre, University of Karachi, Pakistan and accordingly stored at 5-10°C. The nematodes from soil was extracted by means of 3Cobb’s sieving and decanting method (Cobb, 1918) and further purified by Baermann’s funnel technique (Baermann, 1917). The extracted nematodes were quantified in a chamber within 5 ml suspension through counter under the binocular microscope and the same procedure was repeated 3 times and the average reading result found the number of nematodes as per unit of soil sample. Retrieved nematodes were killed instantaneously by pouring a hot water (80-90°C) in a glass cavity block and immediately preserve in 4TAF (Tri- ethanol Amine Formalin) solution for 24 hours (Courtney, Polley and Miller, 1955). After that fixed nematodes were sanitized thrice with distilled water. Specimens were kept for 5-6 days in an incubator at 55°C in 2 ml of 1.25 % glycerin for slow dehydration (Seinhorst, 1959). Permanent mounting was done by transferring of nematodes to a clean microscopic glass slide having pure drop of glycerin and sealed with paraffin wax by gently heating the slide. Later on, measurements were undertaken by de Man’s, 1884 formula through compound microscope Nikon Eclipse E-400. Data Analysis: The aim of area explored in District Lakki Marwat was to ascertain the existence and percentage of Aphelenchid nematodes. The occurrence percentage and relative frequency of nematodes was assessed as follows: Occurence (%) = Total number of positive samples Total number of Samples × 100 10Frequency of Genus/ Species in a sample Relative Frequency (%) = Sum of frequencies of all genus/ species present × 100 Statistically, it was also analyzed by dendrogram of cluster analysis and Simpson diversity index (Simpson, 1949). Diversity index was determined by using the formula as follows: 7∑𝑛(𝑛 − 1) 𝐷 = 1 − 𝑁(𝑁 − 1) D=Diversity, N=Total number of individuals (Genus), n=Number of each individual (Particular Genus). RESULTS During the study of nematodes belonging to the order of Aphelenchida, it was revealed that various locations of district had large number of plant-parasitic nematodes genera including Ditylenchus, Tylenchorhynchus, Pratylenchus, Hoplolaimus, Helicotylenchus, root knot nematodes and other free-living soil nematodes. However, special focus was given only on plant parasitic Aphelenchus and Aphelenchoides due scope of work. Occurrence perecentage: 30 soil smaples out of 61 were found positive and detailed analysis of over all samples revealed two types of genera i.e Aphelenchus (49%) as well as Aphelenchoides (51%). In term of overall occurrence percentage of both genera reported from each site alongwith Globle Positioning System (GPS) was illustrated (Table 1) and earmarked the highest abundance occurrence of both genera in Aba Khel (50% each) and the lowest occurrence in the village Aghzar Khel Aphelenchus (22.7%) and Aphelenchoides (18.1%). Furthermore, occurrence of genus was further probed to ascertain the fact and figure of each genus according to locations where surfaced through applied Relative Frequency (RF). Dendrogram: The dendogram of cluster analysis among the various sites of surveyed areas was applied and accordingly presenting (Figure 1). These locations were divided into two main clades. Sarai Naurang and Tajori has been placed in clade I; while, Aba Khel and Aghzar Khel formed clade II. In the same context, cluster analysis of six nematodes species including Aphelenchus avenae and Aphelenchoides richardsoni formed clade I; while, Aphelenchus isomerus, Aphelenchoides bicaudatus, Aphelenchoides macrospica and Aphelenchoides unisexus form clade II (Figure 2). Simpson Diversity Index: In the same context, diversity index was ensured in ture letter and spirit on all four surveyed sites and resultantly all areas had approximately similar diversity i.e 0.50 as analyzed (Table 2). It has been assessed that environment of district Lakki Marwat is not quite stressfull nor more ecological niches. However, food webs in the areas are relatively simple and does not contain any complexity of food. Absolutely, any changes in the environemt is more likely to be damaging the ecosystem. Composition of Aphelenchus and Aphelenchoides species: During the study two species of Aphelenchus i.e Aphelenchus avenae (Bastian, 1865), Aphelenchus isomerus Anderson and Hooper, 1980 (Ebsary, 1991) and four species of Aphelenchoides comprising Aphelenchoies 16bicaudatus (Imamura, 1931) Filipjev and Schuurmans Stekhoven, 1941, Aphelenchoides richardsoni (Grewal et al., 1992), Aphelenchoides unisexus (Jain and Singh, 1984) and Aphelenchoides macrospica (Golhasan et al., 2017) were identified. It is pertinent to mention herewith that the genus Seinura could not ascertained from district Lakki Marwat. The Aphelenchus avenae was found in highest frequency (40%) from all areas, the second highest Aphelenchoides richardsoni (20%) and followed by the lowest all remaining species (10%) and the same also evaluated vide (Figure 3). All these species previously encountered from different areas of Pakistan; while current survey result showing presence of species (Table 3) and elaborates their area wise presence (Figure 4). Notably, Aphelenchoides macrospica was surfaced for the first time in Pakistan during the research work. DISCUSSION As per previous held record, earlier many species of Aphelenchus of the 11family Aphelenchidae (Fuchs, 1937) Steiner, 1949 and Aphelenchoides of the11family Aphelenchoididae (Skarbilovich, 1947) Paramonov, 1953 were surfaced from case to case research work and accordingly published in several papers from time to time with repsect to numerous hosts, locations and time period from Pakistan. Till to date most of the abundance occurrence specie belonging to the family Aphelenchidae from Pakistan is Aphelenchus avenae )Bastian, 1865(. The said specie has already reported by Anwar and Sarwar, 1981; Gul and Saifullah, 1991 from sample of citrus Spp. Collected from Sargodha, Faisalabad, Sahiwal, Multan, Kalam, Mardan, Peshawar, Mansehra and Hazara. As well as by Khan et al., 2002 found around the root of citrus trees and to evaluate its control by applying indigenous nematicides. Khan, 1991; Maqbool, 1992 from sample of lime collected from district Thatta. Khan and Bilqees, 1985 and Zahid, 2014 found in sample of date palm collected from district Thatta and district Khairpur respectively. Anwar et al., 1986; Maqbool, 1992; Gul and Khan, 2005 found in samples of sugarcane collected from Sargodha, Lahore, Faisalabad, Sahiwal and Charsadda. Khan, 1991; Maqbool, 1992 from lime in Thatta. Gul and Saifullah, 1991; Maqbool, 1988; Saeed et al., 1986; Maqbool, 1986; Maqbool and Zaki, 1992; Maqbool, 1992; Aatika, 2015 from tobaco at Swat, Kalam, Mardan, Mansehra, Abbotabad, Charsada, Hazara, Rawalpindi, Islamabad, Gujrat, Lahore, Sheikhupura and Punjab. Gul and Saifull, 1991; Qasim and Ahmed, 1989; Maqbool, 1988; Maqbool, 1986; Maqbool and Zaki, 1992; Maqbool, 1992; Khan and Hussain, 2004; Bhutta, 2008; Aatika, 2015 from potato samples collected from various places including 8Hunza, Kalam, Peshawar, Kaghan Valley, Bannu, D I Khan, Mansehra, Abbotabad, Hazara, Gujrat, Gujranwala, Faisalabad, Therparker, Hyderabad, Thatta, Karachi, Northern areas and Punjab. Solangi, 1981 found Aphelenchus and Aphelenchoides population in different region of district Hyderabad on the basis of symptoms appearance. Gul and Saifullah, 1991; Maqbool, 1988; Maqbool, 1992; Aatika, 2015 from maize sample collected from Kalam, Peshawar, Mansehra, Abbotabd, Hazara, Sargodha, Mianwali, Sibbi, Chagi, Nawab Shah, Hyderabad, Thatta and Punjab. Maqbool, 1988 and Maqbool, 1992; Aatika, 2015 from sample of wheat collected from Sailkot, Gujranwala, Sheikhupura, Bahawalpur, Rahim Yar Khan, Sukkar, Nawab Shah, Hyderabad and Punjab. Aatika, 2015 found in sample of sorgham collected from Punjab. Maqbool, 1988; Maqbool, 1992 from grapes sample retrieved from Quetta, Pishin and Chagi. Islam et al., 1994; 1996 from sample of apple retrieved from Sawat. Khan and Bilqees, 1994; Khan and Shaukat, 1999; Maqbool, 1988; Maqbool, 1992 from rice sample collected from Bajore Agency, Thatta, Larkana, Nawab Shah and Sanghar. Nematode community of Aphelenchus avenae associated with two rice varieties namely Lateefy and Kharai Ganja were investigated in district Thatta by Khan et al., 2000. Khan et al., 1998 studied the effect of neem cake, neem seed coat and carbofuran on the population density of four nematodes including Helicotylenchus indicus, Merlinus brevidence, Aphelenchus avenae and Meleidogyne Sp. larvae from garlic. Khan et al., 2007; Khan, 2008 from sample of papaya collected from Karachi and Hyderabad. Khan, 2008 from sample of mango collected from Karachi and Hyderabad. Brown, 1962 described Aphelenchus sp from sample of chilli collected from Malir, Karachi, while Khan et al., 2000; Anwar et al., 2013 and Zarina et al., 2015; Aatika, 2015 reported from sample of chilli collected from lower Sindh, Faisalabad, Jhang, Kasur, Lahore, Nawab Shah, Sargodha, T.T. Singh, Sheikhpura, Karachi and Punjab. Nasira et al., 2008 from sample of Ashok tree due to caused of great damges to the tree at Karachi. Anwar et al., 2013 found from sample of tomato collected from Faisalabad, Jhang, Kasur, Lahore, Nawab Shah, Sargodha, T T Singh and Sheikhpura as well as by Samina and Erum, 2019 from Kurrum Agency. Nadia, 2019 from walnut sample collected from Hazara Division. Anwar et al., 2013 from cucumber sample collected from Faisalabad, Jhang, Kasur, Lahore, Nawab Shah, Sargodha, T T Singh, Sheikhpura. Anwar et al., 2013 from sample of bell peppers collected from Faisalabad, Jhang, Kasur, Lahore, Nawab Shah, Sargodha, T T Singh, Sheikhpura. Israr, 2017 reported from radish, turnip and carrot samples collected from KPK and Sindh while on radish, turnip, carrot and sugar beet sample from Punjab. Tariq, 2013 reported Aphelenchus avenae from sample of different medicinal plants including kachnar, jungli badam, aloe, lemongrass, basil, sukhchain and castor collected from Karachi, Thatta and Hyderabad region. Similary, Aphelenchus isomerus Anderson and Hooper, 1980 (Ebsary,1991) by Akhter and Zarina, 2014 found from chilli sample collected from Karachi. In the same way Aphelenchoies bicaudatus (Imamura, 1931) Filipjev and Schuurmans Stekhoven, 1941 by Maqbool, 1986; 1988; 1990; 1992; Maqbool and Zaki, 1992 from sample of rice, kidney bean, pearl millet, sugarcane and maize collected from Sukkar, Nawab Shah, Thatta, Sanghar, Larkana, Dadu, Badin and Khairpur. Shahina and Maqbool, 1992 from sample of banana collected from Sukkar, Nawab Shah and Thatta. Mussarat, 2011 reported Aphelenchus bicaudatus from sample of rice and soybean collected from Faisalabad, Multan, Raheem Yar Khan, Sanghar, Badin and Dir. Israr et al., 2017 from sample of radish, turnip and carrot collected by from Faisalabad, Tando Allahyar, Swat, Sargodha, Dadu, Peshwar, Sialkot, Badin and D. I Khan. Accordingly, in the same context, Aphelenchoides richardsoni )Grewal et al., 1992( from pumpkin and Aphelenchoides unisexus )Jain and Singh, 1984( from tomato sample by Samina and Erum, 2019 collected from Kurram Agency. These genera have been causing serious demages to the root hairs and epidermal tissues which resultantly minize the capability of roots to sucks water and nutrient from the nearby soil (Endo, 1975). Notwithstanding above, the research work was carried out with an aim to get maximum information about presence of nematodes especially Order Aphelenchida in those areas where no research work has been carried out yet. The presence of reported species Aphelenchoides macrospica is an indicator of existence of other undiscovered species. Further research work in other parts of Pakistan will surely be helpful in identifying additional species of nematodes of Aphelenchida. The evidence on nematodes occurrence may be assisted the formers for planning and directing nematode management policies to handle the nematodes population below their threshold levels. Authors Contributions: SK executed survey, collected the data and wrote the manuscript; while SJ supervised the research and reviewed the manuscript. TAK analyzed 6the data. All authors read and approved the final manuscript. Conflict of Interest: The authors declare no conflict of interest.