Proceedings of the Third Academic SessionsA preliminary study on root-knot nematodes, Meloldogyne species and their bacterialhyper-parasite, Pasteuria penetrans associated with Spinach in Matara District.

W.T.S.D. Premachandra'", A.H.P. Lasanthi', K.H.M.A. Deepananda' and R.C. Jayasinghe''.'Department of Zoology, University of Ruhuna, Matara: ZDepartment of Fisheries and Aquatic Resources, Districtoffice, Matara: 'Deparhnent of Agriculture, District office, Matara., *Author for correspondenceE-mail: darnmini0:zoo.ruh.ac.lk. darnminipremachandra@yahoo.co.uk

AbstractA survey was undertaken to investigate, the species composition and distribution of Me 10idogyn enematodes and the prevalence of Pasteuria penetrans, a group of bacterial hyper-parasite ofMeloidogyne nematodes, associated with spinach in Matara district. A total of 11 localities, i.e.,Dandeniya, Denipitiya, Diyalape, Kirimatimulla. Malitnbada, Malara, Mirisisa, Pasgoda,Radampola, Talalla and Welipitiya representing spinach growing regions were selected for thesurvey. At least 10 root systems of mature spinach plants were sampled from each locality. Rootswere assessed for the percentage of root system galled and a gall index (0-5) was assigned. AIleast 20 mature females were checked for the species identification and infection of P. penetrans.Species identification was based on the perineal pattern morphology.Meloidogyne nematodes were recovered from the roots of spinach crops grown at 9 localities,i.e., except, Radampola and Diyalape. Three Meloidogyne species, i.e., M incognita. M.javanica and M arenaria were detected. However, M. incognita and M javanica were foundwith the highest frequencies of occurrence, i.e., 88%for each, while M. arenaria was found in alow frequency, i.e., 2%. In general, mixed populations were detected. Associations of M.incognita and M javanica were common. Sometimes, M javanica, M incognita and M. arenariaoccurred together. Only at one locality, i.e., Talalla, single population, i.e., M javanica, wasprevalent. About 16% of the individual root systems inspected showed more than 50% (gallindex: 5 and 6) of galling while 43% of the roots showed 26-50% (gall index: 4) and theremaining 41% showed 1- 25% (gall index: 1, 2 and 3) galling. Pasteuria penetrans wasrecovered from only three localities, Welipitiya, Denipitiya and Malimbada with the frequenciesof occurrence, 12%, 85% and 4%, respectively. This survey showed that M incognita and Mjavanica were predominant Meloidogyne species associated with spinach in Matara district.

IntroductionSpinach (Spinacia oleracea) is an economically important leafy vegetable crop in Sri Lanka. It iscultivated in small-scale farms and backyard gardens in southern Sri Lanka. Apart from theirimportance as food, it earns cash for the farmers who sell their products in urban areas. Severalplant-parasitic nematodes have been reported on spinach and among these, root-knot nematodes,Meloidogyne spp. are one of the most common and damaging nematodes (Ekanayake, & Toida,1997; Castillo & Jimenez-Diaz, 2003). The primary symptom of root-knot nematode infection isthe formation of typical root galls on the roots. Nutrient and water uptake are reduced because ofthe damaged root system, resulting in weak and poor yielding plants (Abad et al., 2003).Pasteuria penetrans (Thome) Sayre and Starr is an obligate spore-forming group of bacterialparasite of several genera of plant parasitic nematodes (Sturhan, 1988). Only the spore stage isexposed to the soil environment and Meloidogyne nematodes become infected when endosporesof P. penetrans adhere to the cuticle of infective juveniles (Ils) in the soil. Once Us invade theroots and initiate feeding, germination of the spores starts (Sayre & Wergin, 1977). Pasteuriapenetrans has a high potential for reducing Meloidogyne populations (Stirling, 1984; Gowen &Tzortzakakis, 1994) via destroying the reproductive capacity of females (Mankau, 1980) andpreventing invasion of roots by Us (Mankau & Prasad, 1977; Stirling 1984). Several studiesconfirmed prevalence of P. penetrans in Sri Lanka, too (Silva et al., 1996; Mohotti et al., 1998,Mohotti et al., 2002). Meloidogyne infestations often overlooked by growers and confused withnutrient deficiencies and other causes. Most of the vegetable growers in southern Sri Lanka arenot aware of nematode damage on their crops and as a result it goes unnoticed in their fields. Todate, only investigations have been conducted on nematodes associated with vegetable crops inthe southern Sri Lanka. Hence, knowledge on Meloidogyne species composition, damage levels,distribution as well as their natural enemies, associated with vegetable crops including spinach isstill fragmentary. This information is vital to establish successful management practices againstthese nematodes. Therefore, the primary objectives of this study was to investigate the species

127

Proceedings of the Third Academic Sessionscomposition and distribution of Meloidogyne nematodes associated with spinach in Mataradistrict of southern Sri Lanka by means of a survey and occurrence of infections of P. penetranson Meloidogyne species. This study was conducted 'as part of an extensive study of investigatingMeloidogyne nematodes associated with vegetable production in Matara district.

MethodolgyA total of 11 localities, Dandeniya, Denipitiya, Diyalape, Kirimatimulla, Malimbada, Matara,Mirisisa, Pasgoda, Talalla, Radampola and Welipitiya representing spinach growing regions weresampled during June - November 2005 (Figure 1). Spinach crops were sampled from both smallscale farms and home gardens. Ten percent of the total mature spinach crops at each localitywere checked for the presence of root galls based on the visual symptoms, i.e., plants showingchlorosis, wilting and poor growth, or at random where no symptoms were evident. At least 10root systems of these plants were randomly selected and washed to free from adhering soil.Subsequently, the galls per root system were rated and an index of 0-5 (0-5 scale: 0 = no galls; 1= 1-2; 2 = 3-10; 3 = 11-30; 4 = 31-100; 5 = >100 galls) was assigned (Taylor & Sasser, 1978).At least 20 mature females were isolated from each root system using a binocular microscope,examined for P. penetrans infection and number of infected females recorded. The infectedfemales could be identified by the milky white colour and absence of reproductive system and/oregg masses. Infection was confirmed by the presence of cup shaped spores in infected femalesusing a under stereomicroscope at x 400 magnification. Meloidogyne nematodes were identifiedup to the species level using the perennial pattern morphology of females (Eisenback, 1985).

Results and DiscussionMeloidogyne nematodes were recovered from the roots of spinach crops grown at 9 localities,i.e., except, Radampola and Diyalape, in Matara district. At 6 localities, 100% of the spinachplants sampled showed Meloidogyne infestations (Table 1). About 16% of the individual rootsystems inspected showed more than 50% (gall index: 5 and 6) of galling while 43% of the rootsshowed 26-50% (gall index: 4) and the remaining 41% showed 1-25% (gall index: 1, 2 and 3) ofgalling (Table 1). Three species, i.e., Meloidogyne incognita (Kofoid and White) Chitwood,Meloidogyne javanica (Treub) Chitwood and Meloidogyne arenaria (Neal) Chitwood weredetected (Table 1). Meloidogyne incognita was recovered from 8 localities while M javanicawas recovered from 7 localities. In contrast, M arenaria was detected only at 4 localities (Table1 & Figure 2). These findings revealed that M incognita and M javanica were the widelydistributed species on spinach in Matara district while M arenaria occurred only occasionally.In a previous survey, Ekanayake & Toida (1997) reported four most common Meloidogynespecies, i.e., M incognita, M javanica, M. arenaria and Meloidogyne hapla Chitwood mainly onvegetable crops in Sri Lanka. In the same investigation, authors reported M javanica and Mincognita on spinach. In our study, in addition to Mi javanica and M incognita, M arenaria wasrecovered on spinach. "Meloidogyne incognita, M javanica, M arenaria and M hapla have beenrecorded as the most damaging Meloidogyne species in many countries (Olthof & Potter, 1973;Castillo & Jimenez-Diaz, 2003). Lamberti et al., (1987) reported that M incognita was notrecovered from in southern Sri Lanka including Matara district. In contrast, later, Ekanayake andToida (1997) reported M incognita and 1\1. arenaria in Matara district, but notM javanica.When averaged over 9 sampling localities, M incognita and Mi javanica were found with thehighest frequencies of occurrence, i.e., 88% for each, while M arenaria was found in a lowfrequency, i.e., 2% (Figure 2). In general, mixed populations of Meloidogyne nematodes weredetected. Associations of M incognita and M javanica were common and it was obvious that allthe times M incognita dominated M javanica (Figure 2). Sometimes, M javanica, M incognitaand M arenaria occurred together. Associations of M incognita and M arenaria were detectedrarely. At one locality, i.e., Talalla, only M javanica, was prevalent (Figure 2). Lamberti et al.(1987) reported that M arenaria was found only occasionally in many crops includingvegetables and in most cases in mixed populations either with M incognita or M javanica in SriLanka, corroborating present findings.Pasteuria penetrans was recovered only from three localities, Welipitiya, Denipitiya andMalimbada with the frequencies of occurrence, 12%, 85% and 4%, respectively. Mohotti et al,(1998) reported that there is a natural suppression of plant parasitic nematodes by P. penetransgroup in tea plantations in Sri Lanka. Moreover, P. penetrans has been isolated from

128

Proceedings of the Third Academic SessionsMe/oidogyne nematodes associated with tomatoes in Matara district (Silva et al., 1996).Our findings showed that M incognita and M javanica were predominant species associatedwith spinach in Matara district. The reasons for the 'absence of Meloidogyne infestations atRadompola and Diyalape were most probably associated with application of Carbofuran to thesoil prior to planting and heavy application of poultry manure to the soil, respectively. The dataon percentage root galling indicated that these nematodes have a high potential to affect growthof the spinach crops. Prevalence of P. penetrans in some cultivation areas showed potential signsof natural management of Meloidogyne nematodes which could be used in integratedmanagement strategies.

ReferencesAbad, P., Favery, B., Rosso, M-N., and Castagnone-Serena, P. (2003). Root-knot nematode parasitism and host

response: molecular basis of a sophisticated interaction. Molecular Plant Pathology 4,217-224.Castillo, P., and Jimenez-Diaz R.M, (2003). First report of Meloidogyne incognita infecting spinach in Southern Spain.

Phytopathology 87,874 (Abstract).Eisenback, J.D. (1985). Diagnostic characters useful in the identification of the four most common species of root-knot

nematodes. In An Advanced Treatise on Meloidogyne Vol II Methodology. (K.B. Barker, C.C. Carter andJ.N. Sasser, eds). North Carolina State University, NC, U.S.A, pp 95-102.

Ekanayake, H. M. R. K., and Toida, Y. (1997). Nematode parasites of Agricultural Crops and their distribution in SriLanka. JIRCAS Journal 4, 23-39.

Gowen, S.R., and Tzortzakakis, E.A. (1994). Biological control of Meloidogyne species with Pasteuria penetrans.Bulletin OEPPIEPPO 24,495-500.

Lamberti, F., Ekanayake, H. M. R. K., and Di Vito, M. (1987). The root-knot nematodes Meloidogyne species foundin Sri Lanka FAO. Plnat Protection Bulletin 35, 163-166.

Mankau, R. (1980). Biological control of Meloidogyne populations by Bacillus penetrans in West Africa. Journal ofNematology 12,230 (Abstract).

Mankau, R., and Prasad, N. (1977). Infectivity of Bacillus penetrans in plant parasitic nematodes. Journal ofNematology 9, 13-15.

Mohotti, K.M., Bridge J., and Gowen, S.R. (1998). Natural suppression of plant parasitic nematodes in tea soils in SriLanka. (Abstr.). Abstracts of the 24th International Symposium of the European Society ofNematologists, 4-9th August, 1998, Dundee, Scotland, pp.73.

Mohotti. K.M., Gowen, S.R., and Bridge, J. (2002). Morphological and morphometric characteristics of Sri Lankanpopulations of mature endospores Pasteuria. Fourth International Congress of Nematology Programme andAbstracts, June 2002, Tenbel, La Galletas, Arona, Tenerife, Canary Islands, Spain, pp. 8-13.

Olthof T.H.A and Potter J.W. (1973). The relationship between population densities of Pratylenchus penetrans andcrop losses in summer-maturing vegetables in Ontario. Phytopathology 63,577-582.

Sayre, R.M., and Wergin, W.P. (1977). Bacterial parasite of a plant nematodes; morphology and ultrastructure.Journal of Bacteriology 129, 1091-1101.

Silva, M.P. De., Premachandra, D., and Gowen, S.R. (1996). Studies on the detachment of spores of Pasteuriapenetrans attached to its host Meloidogyne javanica. Afro-Asian Journal of Nematology, 6, 110-113.

Stirling, G.R. (1984). Biological control of Meloidogynejavanica with Bacillus penetrans. Phytopathology 74,458-462.

Sturhan, D. (1988). New hosts and geographical records of nematode parasitic bacteria of the Pasteuria penetransgroup. Nematologica, 34, 350-356.

Taylor, A.L., and Sasser, J.N.' (1978). Biology, identification and control of root-knot nematodes (Meloidogynespecies). North Carolina State University Graphic, Raleigh NC (U.S.A), pp. Ill.

Figure 1: Sampling sites

129

Proceedings of the Third Academic Sessionsc=:J MeIokbgyne Inrognita=Me~javanic8_ MeIoIdogyne arenarlaI100

§ 90~ 80

J :I:I30

i 20

$ 10

j 0~ 8 iii

Locality

Figure 1. Mean (± SE) percentage frequency of occurrence ofthree Meloidogyne species associated with spinach in IIdifferent localities in Matara district.

Table 1. Number of spinach plants checked for the presence of root galls, percentage plants with root galls, averageroot gall index and Meloidogyne species associated with roots of spinach at II localities in Matara district.

Locality No. of Percentage plants Average Meloidogyne species foundplants* with root galls root gallchecked index

Dandeniya 6 100 4Denipitiya 45 78 3Diyalape 6 0Kirimatimulla 10 100Malimbada 12 100Matara 10 100Mirisisa 50 16Pasgoda 8 25Radampola 6 0Talalla 15 100 4 M.javanicaWelipitiya 10 100 4 M. incognita, M.javanica, M. arenaria

*10% of the total mature spinach crops at each locality were checked for the presence of rootgalls.

M incognita, M javanica, M arenariaM incognita, M javanica

3 M incognita, A1.javanica!V!. incognita, .\1. javanica, M arenariaM incognita, M. javanicaM. incognita, M javanicaM. incognita, !vi arenaria

543

130