Exploring Citrus Nematodes and its Antagonists in Citrus Growing Areas of Punjab, Pakistan

Muhammad Nadeem Asghar, Sajid Aleem Khan, Nazir Javed, Muhammad Arshad


Citrus nematode (Tylenchulus semipenetrans) is a major threat to the citrus industry in Pakistan. During 2019-20, a survey was carried out for citrus nematode and its antagonists in citrus orchards (Both healthy and declining trees were samples) of Punjab, Pakistan. Bhalwal, Shahpur, Sillanwali, Kotmomin, Sargodha, Quaidabad, Jhang, and Rahim Yar Khan were surveyed for disease incidence, prevalence, and association of T. semipenetrans. Maximum disease prevalence (100%) was recorded in Rahim Yar Khan while minimum (20%) in Bhalwal and Kotmomin. Maximum slow decline with 80% field incidence was observed in Rahim Yar Khan while a minimum of 20% in Quaidabad.  Soil and root samples were used for nematode extraction using Baerman's funnel method and the Whitehead Hemming tray method. A maximum number of J2s/100ml of soil were observed in soil samples taken from Rahim Yar Khan (1674) followed by Shahpur (1534), Qaidabad (1432), Sargodha (1347), and Bhalwal (1172). The highest number of nematode females per gram of root were recorded in Rahim Yar Khan (652) followed by Quaidabad (611), Bhalwal (490), Kotmomin (421), Sillanwali (387). Isolation of different fungal and bacterial antagonist isolates was also done by soil dilution plate technique using nutrient agar media and potato dextrose agar media. Identification of fungal antagonists was made on colony growth, color, and spore structure. Bacterial identification was also done on morphological characters and gram tests. Trichoderma harzianum, Trichoderma viride, Trichoderma koningii Trichoderma atroviride, Pseudomonas fluorescens, Pseudomonas putida, Bacillus cereus and Bacillus subtilis were found associated.


Slow Decline; Citrus Nematode; Prevalence; Incidence; Trichoderma; Pseudomonas; Bacillus

Full Text:



Abd-Elgawad, M.M. 1992. Spatial distribution of the phytonematode community in Egyptian citrus groves. Journal of Fundamental and Applied Nematological Research. 15: 367-373.

Abd-Elgawad, M.M., F.A. Al-Yahya, and Z.A. Stephan. 2010. [Nematodes of citrus.] In: Abu-Gharbieh, W.A., A.S. Al-Hazmi, Z.A. Stephan & A.A. Dawabah (Eds). [Plantnematodes in Arab countries.] Amman, Jordan, Arab Society of Plant Protection, pp. 553-602.

Ahmad, M.S., T. Mukhtar and R. Ahmad. 2004. Some Studies on the Control of Citrus Nematode (Tylenchulus semipenetrans) by Leaf. Asian Journal of Plant Sciences. 3(5):544-548.

Ahmad, R. and H. Khan. 1999. Citrus slow decline: Problems and progress in the Punjab. Natl. Conference of Plant Pathol, Sept. 27-29. Univ. Agric. Faisalabad: 20-23.

Ahmed, W., M.A. Nawaz, M.A. Iqbal and M.M. Khan. 2007. Effect of different rootstocks on plant nutrient status and yield in Kinnow mandarin (Citrus reticulata Blanco). Pakistan Journal of Botany. 39(5):1779-1786.

Al-Azzeh, T. and W. A. Gharbieh. 2005. Factors related to seasonal changes in numbers of Tylenchulus semipenetrans in the Jordan valley. Pakistan Journal of Nematology. 23(2):241-250.

BacDive. 2021. Explore Bacterial Diversity. Online bacterial diversity metadatabase. Leibniz Institute DSMZ, Inhoffenstraße 7B, 38124 Braunschweig, Germany.

Bakker, P.A., C.M. Pieterse and L.C. Van Loon. 2007. Induced systemic resistance by Pseudomonas fluorescens spp. Phytopathology. 97(2):239-243.

Brown, K. F. 1962. A survey of plant parasitic nematodes problem in Pakistan. Shell Intl. Che. Co. Ltd. 9p.

Byrd Jr. D. W., T. Kirkpatrick and K. R. Barker. 1983. An improved technique for clearing and staining plant tissue for detection of nematodes. Journal of Nematology. 15(1):142-143.

Chohan, R. A., S. A. Anwar and N. Javed. 2007. Prevalence of citrus nematode, Tylenchulus semipenetrans in four citrus producing areas of Punjab. Int. Symp. On prospects of Hort. Ind. In Pak. Ins. Hort. Sci., Univ. Agri., Faisalabad. 159p.

Diwan, S. N. H., R. A. Al-Ani and M.A. Adhab. 2011. Systemic resistance induced in potato plants against Potato Virus Y common strain (PVY) by plant extracts in Iraq. International Journal of Environmental Sciences and Technology. 5(1): 209-215.

Ducan, L. W. and W.N. Jo. 1987. The relationship between development of the citrus root system and infestation by Tylenchulus semipenetrans. Revue Nematology. 10 (1):61-66.

FAO. (United Nations, New York, United States, 2019).

FOF. 2021. Faces of Fungi, Fungal Phyla. The Mushroom Research Foundation. Chiang Rai, Thailand.

Gamalero, E., & Glick, B. R. (2020). The Use of Plant Growth-Promoting Bacteria to Prevent Nematode Damage to Plants. Biology, 9(11), 381.

Gene, J., l. S. Verdejo, A. M. Stchigel, F. J. Soribass and J. Guarro. 2005. Microbial parasites associated with Tylenchulus semipenetrans in citrus orchards of Catalonia, Spain. Biocontrol Science and Technology. 15(7):721-731.

Hammam, M. M. A., W. M. El-Nagdi, and M.M.M. Abd-Elgawad. 2016. Biological and chemical control of the citrus nematode, Tylenchulus semipenetrans (Cobb, 1913) on Mandarin in Egypt. Egyptian Journal of Biological Pest Control. 26 (2):345-349.

Harman, G.E., C. R. Howell. Viterbo I. Chet and M. Lorito. 2004. Trichoderma species - opportunistic, avirulent plant symbionts. Nature Reviews Microbiology. 2(1):43-56.

Heald, C.M. and J.H. O Bannon. 1987. Citrus decline caused by nematodes. V. Slow decline. Florida Depart. Agric. consumer Servic. Div. plant industry, Nematology. Circular. 143:4.

Howell, C.R. 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts. Plant Disease. 87(1):4-10.

Hussain, M. A., T. Mukhtar, M. Z. Kayani. 2011. Efficacy evaluation of Azadirachta indica, Calotropis procera, Datura stramonium and Tagetes erecta against root- knot nematodes Meloidogyne incognita. Pakistan Journal of Botany. 43:197–204.

Hussain, M.A., I. Fatima, T. Mukhtar, M.N. Aslam and M.Z. Kayani. 2015. Effect of inoculum density of root-knot nematode Meloidogyne incognita on damage potential in eggplant. Journal of Myco- phytopathological Society of Pakistan. 13(1):33-36.

Ibrahim, I.K.A. (1994). Potentially important phytoparasitic nematodes in agroforesty plantations and the associated host plants. 101-109 pp. In: Proc. second Afro-Asian Nematology Symposium. Minufiya, Egypt, December 18-22, 1994.

Iqbal, M. A., T. Mukhtar, R. Ahmad, and H. U. Khan. 2006. Ecological prevalence of Tylenchulus semipenetrans in four districts of the Punjab Province, Pakistan. Pakistan. Journal of Nematology. 24(1):19-26.

Johnson, L. F. and E. A. Curl. 1972. Methods for research on the ecology of soil borne plant pathogens. Burgess Publishing Company Minneapolis, PP 247.B

Li, J., T. C., Todd, J. Lee and H. N. Trick. 2011. Biotechnological application of functional genomics towards plant-parasitic nematode control. Journal of Plant Biotechnology. 9(7):797-806.

Mckerny, M.V. and P. A. Roberts. 1985. Phyto nematology study Guide Cooperative Extension University of California. Division of Agriculture and Natural Resources. 4045. P 56.

Mukhtar, Tariq., Muhammad Tariq-Khan, Naveed Aslam. 2021. Bioefficacy of Trichoderma Species Against Javanese Root-Knot Nematode, Meloidogyne javanica, in Green Gram. Gesunde Pflanzen 73 (3).

Nasir, J., R. Ali, Saifullah and H. U Din. 2021. Incidence of citrus nematode Tylenchulus semipenetrans Cobb in citrus growing areas of Khyber Pakhtunkhwa province of Pakistan and its organic management. Arch. Phytopathology. 12(1):1-15.

Parvatha, R. P., M. S. Rao and M. Nagesh. 1996. Management of citrus nematode, Tylenchulus semipenetrans by integration of Trichoderma harzianum with oil cakes. Nematologia Mediterranea. 24: 265-267.

Parvez, I., T. Mukhtar and M. Faiz. 2003. Occurrence of citrus nematode (Tylenchulus semipenetrans) in Sargodha district. Journal of Research (Sci.). Bahauddin Zakariya Univ. Multan. 14(1):91-95.

Rachel, W. 2021. Summary of biochemical tests used to identify bacteria. General and Medical Microbiology (MOLB/ MICR). The virtual edge, University of Wyoming, University Ave. Laramie, WY.

Rashidifard, M., Shokoohi, E., Hoseinipour, A., & Jamali, S. (2015). Distribution, morphology, seasonal dynamics, and molecular characterization of Tylenchulus semipenetrans from citrus orchards in southern Iran. Biologia, 70(6), 771-781.

Rehman, A., M. Saleem, S. Mehboob and A. A. Bokhari. 2011. Fungi associated with rhizosphere soil in mango decline orchards and their in vitro control. Pakistan Journal of Phytopathology. 23(2):112-117.

Safdar, A., N. Javed, S. A. Khan, H. U. Khan, A. Rehman and I. U. Haq. 2010. Survey and investigation of different citrus growing areas for citrus sudden death syndrome. Pakistan Journal Phytopathology. 22(2):71-78.

Schaad, N. W., J. B. Jones and W. Chun. 2001. Laboratory guide for the identification of plant pathogenic bacteria. (3rd Ed.). American Phytopathological Society (APS Press).

Shah, M. A. 2004. Citrus cultivation in NWFP. p. 39-39. Proc. Int. Conf. Citriculture University of Agriculture, Faisalabad.

Sharma, P. and R. Pandey. 2009. Biological control of root-knot nematode; Meloidogyne incognita in the medicinal plant; Withania somnifera and the effect of biocontrol agents on plant growth. African Journal of Agricultural Research. 4(6):564-567.

Sharon, E., M. Bar-Eyal, I. Chet, A. Herra- Estrella, O. Kleifeld, and Y. Spiegel. 2001. Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum. Phytopathology. 91:687-693.

Singh, V., R. Mawar, and S. Lodha, S. 2012. Combined effects of biocontrol agents and soil amendments on soil microbial populations, plant growth and incidence of charcoal rot of cowpea and wilt of cumin. Phytopathologia Mediterranea. 51:307–316.

Suganthi, K., P. Vetrivelkalai, K. Poornima and R. M. Vijayakumar. 2019. In vitro bioefficacy of endophytic isolates against citrus nematode, Tylenchulus semipenetrans. International Journal of Current Microbiology and Applied Sciences. 8(5):1050-1055.

Verdejo-Lucas, S. and M.V. Mckenry. 2004. Management of the citrus nematode Tylenchulus semipenetrans in citrus. Journal of Nematology. 36(4):424-432.

Whitehead, A. G. and A. K. Hemming. 1965. Comparison of quantitative method of extracting the small vermiform nematode from soil. Annals of Applied Biology. 55: 25-38.

Zadoks JC, Schein RD. 1979. Epidemiology and Plant Disease Management. New York: Oxford University Press.

DOI: https://doi.org/10.33687/jpe.003.02.3714


  • There are currently no refbacks.

Copyright (c) 2021 Muhammad Nadeem Asghar, Sajid Aleem Khan, Nazir Javed, Muhammad Arshad

Journal of Plant and Environment
ISSN: 2710-1665 (Online), 2710-1657 (Print)
© EScience Press. All Rights Reserved.