Citrus limon (L.) is a tree with evergreen leaves and yellow edible fruits belong to the Family-Rutaceae. It is also known as lemon (English), Le citron (French), Zitrone (German), limón (Spanish) and níngméng (Chinese).

Citrus is the third largest fruit crop which is grown commercially in large areas of Rajasthan, Maharashtra, Andhra Pradesh, Karnataka, West Bengal, Sikkim, Punjab and Assam in arid & semi-arid regions to humid tropical regions of India. Presently, area under citrus is 1.09 mha with a production of 14.50 million tonnes and average productivity of 11.80 tonnes/ha in India (Agristat, 2020). The most important commercial citrus species in India are the mandarin orange (C. reticulata), sweet orange (C. sinensis), acid lime (C. aurantifolia) & lemon (C. limon) sharing production 28.36, 44.29 and 27.34 percent, respectively. In Rajasthan, lime & lemon occupy an area of 2.69 thousand ha with 14.47 thousand MT production and productivity of 5.66 tonnes/ha (Horticulture Statistics at a Glance, 2018). Nutritional facts of citrus per 100 g is Calories 60, Fat N/A, Sodium N/A, Fiber 3 g, Sugar 12 g, Protein 1g, Vitamin A 14 mcg and Vitamin C 70 mcg. In C. limon seed oil, the main ingredients are fatty acids, such as arachidonic acid, behenic acid and linoleic acid and also tocopherols and carotenoids (Malacrida et al., 2012; Mucci et al., 2013). The main components of the C. limon essential oil are monoterpenoids. In addition to terpenoids, the essential oil also contains linear furanocoumarins (psoralens) and polymethoxylated flavones (Russo et al., 2015; Kaskoos, 2019; González-Molina et al., 2010). Analysis of macro elements in C. limon fruit showed the presence in pulp and peel of calcium, magnesium, phosphorus, potassium and sodium (Czech et al., 2020).

Lemon fruit stands out as having well-known nutritional properties, its valuable biological activities are underestimated in modern phytotherapy and cosmetology (Goetz, 2014). It is extensively used in the food, pharmacy and cosmetics industries etc. (Abad-García et al., 2012; García-Salas et al., 2013). Lemon juice has traditionally been used as a remedy for scurvy disease of human system.

An alarming situation in citrus cultivation has developed due to dwindling of the area under citrus as it is of late threatened with the serious disease commonly known as "Citrus decline". It is well known that citrus decline is a complex disease. Citrus crop is attacked by number of insect pests (Citrus aphid, Lemon butterfly, Citrus psylla, mealy bug, leaf minor and fruit sucking moth) and pathogens (Citruscanker, scab, citrus tristeza disease, greening, anthracnose and sooty mould) including nematodes. Among nematodes, Tylenchulus semipenetrans Cobb, 1913 is one of the most destructive nematode species associated with citrus (Fig. 1) and is known to occur in all the citrus growing areas of the world (Thorne, 1961; Nasir et al., 2021). The citrus nematode was first discovered in California by Cobb (1913) and placed it in a new genus and named this species Tylenchulus semipenetrans on the basis of position in the root. This nematode is present in about 92 per cent of Southern California citrus orchard and is believed to be one of the important causal factors of the citrus replant problem (Thomas, 1913; Baines and Clark 1952; Abd-Elgawad and Mahfouz 2020). A significant yield drop of 20-30% is caused by the citrus nematode (Philis, 1989; Kumar et al., 2020). In India, the first report on the presence of T. semipenetrans was made by Siddiqi (1961). It is now known to be widely distributed in the country including Punjab, Delhi, Rajasthan, Uttar Pradesh, Maharashtra, Assam, Orissa and Kerala and causing great damage directly or indirectly in citrus (Swarup et al., 1964; Chona et al., 1965). It can be manage by eco-friendly management options may be employed in place of pesticides against citrus nematode, T. semipenetrans on lemon to promote organic and economical cultivation of lemon and INM (Mukesh Jaiman et al., 2024).

The agro-climatic condition of Rajasthan is suitable for the growing Lemon. The citrus nematode, Tylenchulus semipenetrans has been found more frequently associated with lemon (Citrus limon) and which results for severe economic losses. A survey was carried out in Udaipur district to find out occurrence and population status of citrus nematode on lemon. Soil and root samples were collected from surveyed areas and brought to the laboratory for processing. Two hundred centimetre cubic soil were processed for estimation of nematode juveniles by Cobb’s sieving and decantation method followed by Baermann’s funnel technique. Roots were be stained using 0.1 % acid fuchsine lacto phenol solution, rinsed in water to remove excess amount of stain and keep in clear lacto phenol at least for 24 hours before examination (Mc Beth et al., 1941) and different observations were taken viz., number of females/5 g roots and number of egg masses/5 g roots and nematode population/200 cc soil etc. At the time of survey, GPS coordinates (longitude and latitude) were also taken to determinate the geographic location on maps of different localities where samples were collected (Table 1).

A. Occurrence of citrus nematode:

The per cent occurrence of T. semipenetrans was calculated by under given formula

Fig. 1: (1) Healthy and Citrus nematode infested lemon; (2) Severe infection of Tylenchulus semipenetrans on lemon roots (3) Life-Cycle of Citrus nematode, Tylenchulus semipenetrans on Lemon. (3A: Establishment of feeding site by second stage juveniles (J2) of Tylenchulus semipenetrans; 3B: Third stage juvenile; 3C: Pre mature female and fourth stage juvenile of citrus nematode; 3D&E: Mature female buried their mouth in roots and separate embedded female from infected root with damaged mouth parts due to make a permanent feeding site; 3F: Soil dirt particles clinging the roots due to gelatinous matrix which is secreted by female of T. semipenetrans)

Table 1: Name of localities and GPS coordinates of surveyed citrus growing areas in and around Udaipur, Rajasthan.

Fig. 2. GPS locations of surveyed citrus growing areas in and around Udaipur to find out population status of Citrus nematode on lemon.

To ascertain the prevalence and population analysis of the citrus nematode, Tylenchulus semipenetrans, a survey was conducted in the Udaipur district of Rajasthan from July to December 2022. A total of three hundred fifty soil and roots samples were obtained from citrus groves. Soil and roots samples collected, brought to the laboratory and stored them in the refrigerator till processing. After processing the samples using Cobb's sieving and decanting method followed by Baermann’s funnel assembly, the samples were checked for citrus nematode presence using a stereo binocular microscope. Results expressed that the occurrence of T. semipenetrans was observed 71.92 per cent from Udaipur district with an average population of 86.40 females per 5 g of roots, 53.27 egg masses per 5 g of roots and588.46 Nematode per 200 cc soil. Observations on females per 5 g of roots, egg masses per 5 g of roots and nematode population per 200 cc soil were recorded and presented in Table 2 with occurrence per cent and illustrated through Fig. 2 and 3.

1. Girwa. In the Girwa tehsil, three localities were surveyed, which were RCA farm, Kaya and Ramarate. Number of soil and root samples collected from these localities was 20, 10 and 20, respectively. Among them Tylenchulus semipenetrans was recorded at 100%, 80% and 60% occurrence, respectively. Nematode population viz., number of females per 5 g roots was 110.25, 96.20 and 56.00 and number of egg masses per 5 g roots was 76.15, 65.90 and 32.00 and number of nematodes per 200 cc soil was 1050.00, 355.00 and 475.00, respectively from each locality. Highest number of females, egg masses per 5 g roots and nematodes population per 200 cc soil were found in RCA farm within Girwa tehsil with a number of 110.25, 76.15 and 1050.00 with 100% frequency of occurrence.

2. Rishabdev. Ramabaori, a village in the Rishabdev tehsil, was the localities that underwent survey. Ten soil and root samples were taken from the area. Tylenchulus semipenetrans was found in 80% of them. In number, 100.20 females and 72.70 egg masses were found in 5 g of roots. While, the nematode population per 200 cc soil were 400.00.

3. Kherwara. Kherwarakhalsa, a locality in the Kherwara tehsil, was the one that underwent a survey. A total of ten samples of the soil and roots were taken. A 60% occurrence of the citrus nematode was noted among them. There were 69.00 females and 38.20 egg masses per 5 g of roots and the number of nematode in 200 cc soil were 425.00.

4. Vallabhnagar. In the tehsil of Vallabhnagar samples were collected from nearby areas. Ten soil and root samples were collected from the locality. Among them citrus nematode (Tylenchulus semipenetrans) was recorded at 70% occurrence. Number of females per 5 g roots was 80.10 and egg masses per 5 g of roots were 50.50 and nematodes per 200 cc soil was 670.00.

5. Jaisamand. Kewdakhurd, a hamlet in the Jaisamand tehsil, was surveyed. Ten samples of the soil and root system were taken. 70 percent of them were found infested with the citrus nematode Tylenchulus semipenetrans. The ratio of females to 5 g of roots was 76.35. The egg masses per 5 g of roots were 42.40, while the nematode population per 200 cc of soil was 455.00.

6. Sayra. Sakariya and Nandeshma, two settlements in the Sayra tehsil, were surveyed. A total of 20 and 10 samples of soil and roots were taken from each location, respectively. Tylenchulus semipenetrans was found with the occurrence of 80% and 90% of them, respectively. There were 102.00 and 114.30 females per 5 g of roots, respectively and the egg masses per 5 g of roots were 68.00 and 73.10, respectively, while the larval densities per 200 cc of soil were 405.00 and 545.00 from each site. Nandeshma had the highest female populations, egg masses per 5 g roots, and nematode population per 200 cc soil.

7. Gogunda. In the Gogunda tehsil, two localities were surveyed, which were Paaner and Gogunda. Ten soil and root samples were collected from each locality. Out of them citrus nematode (Tylenchulus semipenetrans) was recorded at 90% and 80% occurrence, respectively. Number of females per 5 g roots was 98.60 and 146.70, respectively. Number of egg masses per 5 g roots was 54.90 and 82.40 and nematode population per 200 cc soil was 730.00 and 1355.00 from each locality. Highest females, egg masses per 5 g roots and nematode population per 200 cc soil were found in Gogunda.

8. Jhadol. Eight localities were surveyed in Jhadol tehsil, which were Bansiwara, Rohimala, Biroti, Khakhar, Kolar, Parda, Bhakoomba, and Phalasiya. Numbers of soil and root samples collected from each locality were 20, 10, 20, 10, 10, 10, 10 and 10, respectively. Out of them citrus nematode was recorded at 80%, 70%, 60%, 70%, 50%, 0%, 80% and 70% occurrence, respectively. Number of females / 5g roots was 79.20, 78.80, 98.50, 99.30, 56.40, 0, 110.00 and 67.60, respectively. Egg masses/5g of roots were 41.65, 39.30, 68.80, 65.30, 28.60, 0, 72.00 and 34.20, respectively and nematode population/200 cc soil were 450.00, 375.00, 800.00, 530.00, 295.00, 0, 860.00 and 645.00 from each locality. Highest females, egg masses/ 5g roots and nematode population/ 200 cc soil were found in Bhakoomba.

9. Bhinder. In the tehsil of Bhinder four localities were surveyed, which were Kheroda, Bagar, Amarpura and Gumanpura. Number of soil and root samples collected was 20, 10, 10 and 10 from each locality. Out of them 85%, 80%, 60% and 80% samples were infested with the citrus nematode (Tylenchulus semipenetrans), respectively. Number of females (108.60, 78.30, 95.00 and 116.50) and egg masses (70.35, 44.70, 60.00 and 78.00) per 5 g of roots were, respectively and nematode population per 200 cc soil (925.00, 510.00, 745.00 and 965.00) was recorded from each locality. Highest females, egg masses per 5 g roots and nematode population per 200 cc soil were found in Gumanpura.

10. Mavli. Jawanjika Kheda and Gadariya was were the two locations in the Mavli tehsil that were surveyed. 20 and 10 samples of soil and roots, respectively, were taken from each location. Citrus nematode was found in 65% and 100% of them, respectively. There were 85.20 and 86.70 females per 5 g of roots, respectively. The egg masses per 5 g of roots from each site were 62.30 and 48.20, respectively, while the nematode population per 200 cc soil was 955.00 and 280.00. Jawanjika Kheda had the highest number of females, egg masses per 5 g roots, and nematode population per cc soil.

11. Salumber. In the tehsil of Salumber, one locality was surveyed, which was Seriya. Number of soil and root samples collected was twenty from the locality. Out of them citrus nematode (Tylenchulus semipenetrans) was recorded at 60% occurrence. Number of females (36.60) and egg masses (15.40) per 5 g of roots were recorded and nematode population per 200 cc soil were 100.00.

Table 2: Frequency of occurrence of citrus nematode, Tylenchulus semipenetrans and population status of citrus nematode, Tylenchulus semipenetrans on lemon in and around Udaipur, Rajasthan.

Citrus nematode, Tylenchulus semipenetrans was first reported from California citrus orchards in United States of America by Cobb (1913). Later in 1961 its occurrence was reported in India in Aligarh, Uttar Pradesh. However, in India very little work has been done under survey. To fill this gap, survey was undertaken during the July, 2022 to December, 2022 in and around Udaipur district of Rajasthan. During survey, three hundred and fifty soil and root samples were collected from Udaipur (RCA farm, Kaya, Ramarate, Ramabaori, Kherwarakhalsa, Vallabhnagar, Kewdakhurd, Sakariya, Nandeshma, Paaner, Gogunda, Bansiwara, Rohimala, Biroti, Khakhar, Kolar, Parda, Bhakoomba, Phalasiya, Kheroda, Bagar, Amarpura, Gumanpura, JawanjikaKheda, Gadariyawas, Seriya) district of Rajasthan. Results expressed occurrence of T. semipenetrans 71.92 per cent from surveyed area of Udaipur district. Highest occurrence was observed in RCA & Gadariya was (100%) followed by Nandeshma & Paaner (90%) and Kheroda (85%). Maximum population of females per 5 g roots were recorded from Gogunda (146.70) followed by Gumanpura (116.50) and Nandeshma (114.30). In case of egg masses per 5 g roots, highest egg masses were found from Gogunda (82.40) while 78.00 & 76.15 egg masses per 5 g roots were observed from Gumanpura and RCA, respectively. Further, Nematode per 200 cc soil were found 1355.00, 1050.00 & 965.00 in descending order from Gogunda, RCA farm and Gumanpura, respectively.

Fig. 3. Frequency of Occurrence of citrus nematode, Tylenchulus semipenetrans on lemon in and around Udaipur.

In Parda village, citrus nematode was not noticed. Overall highest nematode infection was found in Gogunda followed by RCA and Gumanpura. The results of present investigation are in accordance with the findings of earlier workers who reported its wide distribution and varied population from India as well as in abroad. Khanzada et al. (2008) recorded the incidence of T. semipenetrans in all citrus varieties varied from 56% for mandarin followed by 40% for lemon & orange and 35% for kinnow & musambi. Bakr et al. (2011) carried out a survey to know the occurrence of T. semipenetrans in Egypt and results revealed occurrence of T. semipenetrans was 85.18 %. Abu Habibet al. (2020) conducted a survey in Alexandria, El-Behera and Kafr El-Shiekh governorates, northern Egypt in citrus groves and reported that the citrus nematode was found to be very common frequency of occurrence (FO) = 100% in all the surveyed locations. Nasir et al. (2021) surveyed citrus orchards at Peshawar, Mardan, Nowshera and Malakand for citrus nematode. Maximum infestation (86.00%) was recorded at Pallai in Malakand division while minimum infestation (44.00%) was recorded in Malakandher, Peshawar. Jabbar and Abedulridah (2023) carried out a survey on T. semipenetrans (Cobb) and found the presence of citrus nematodes infestation in all orchards and in all regions. It was found that the severity of infection was significantly higher with older trees compared to newly established orchards with young plants. Haseeb et al. (2024) carried out a survey for the assessment of nematode population distribution in all 7 Tehsils of district Sargodha (Pakistan) and showed that the population was low in declining orchards as compared to the population densities found in healthy orchards.

The variation in occurrence and citrus nematode population may be due to variation in varieties, age of orchards, soil texture, soil temperature and relative humidity as well as other agro-ecological conditions during survey. Most of the citrus groves in area of Udaipur were infected with the citrus nematode, Tylenchulus semipenetrans with the occurrence of 71.92%. The nematode is responsible to cause slow decline with the symptoms of chlorosis and as well as dead twigs and in case of nursery stage it also reduce the root system drastically and ultimately reduced the quality & quantity of yield.

Mukesh Jaiman  and B.L. Baheti  (2024). Occurrence and Population Status of Citrus Nematode on Lemon in Udaipur, Rajasthan. Biological Forum – An International Journal, 16(10): 165-171.