Australasian Plant Pathology (1998) 27: 84-89
Optimisation of culture conditions for in vitro infection of tomato with the root-knot nematode Meloidogynejavanica P. Hutangura, M.G.K Jones and T. Heinrich WA State Agricultural Biotechnology Centre, Murdoch University, Murdoch, Western Australia 6150 Australia Corresponding author: T. Heinrich (Email
[email protected])
Abstract Conditions for in vitro culture of tomato seedlings (Lycopersicon esculentum) were optimised to yield high in vitro rates of infection by the root-knot nematode Meloidogyne javanica. The influence of nutrient salts, gelling agents, sucrose concentration and pH on the development of nematode-induced root galls was investigated. A quarter-strength Murashige and Skoog medium supplemented by 0.5°;!) sucrose and solidified with 0.6% phytagel at pH 6.4 gave most galls on tomato roots. Eggs were sterilised in 2.5~" sodium hypochlorite for 4 min followed by 0.2% mercuric chloride for 4 min. Surface-sterilised eggs showed a 16% cumulative hatching rate within 10 days. Tomato seedlings cultivated in vitro for 1 week were inoculated with sterile eggs and the infection process was monitored weekly. After 5 to 7 weeks, sterile egg masses were harvested, second-stage juveniles were hatched in sterile distilled water and used to re-inoculate tomato seedlings without further sterilisation. The culture conditions described gave similar infection results for the related root-knot nematodes AI. incognita, M. arenaria and AI. hapla.
Additional keywords: monoxenic culture, nematode sterilisation.
Introduction Root-knot nematodes (Meloidogyne spp.) are important pests which attack most economic crops, causing major yield lossand a decreasein the quality of produce. In 1987, worldwide crop losses caused by root-knot nematodes were estimated at US$77 billion(Sasser andFreckman 1987) andthese were probably underestimated because nematode infectionsare often not detecteduntil rootsand soil are studied (Waitersand Barker 1994). Whenrootknot nematodes parasitise roots, they induce specialisedfeeding structures,called giant cells, in the host tissue. In vitro culture is an advantageous way to examine these feeding structures and the concomitant alterations in plant cell morphology because it allows permanent microscopicobservation of the plant infection process and excludes interaction with other soil pathogens (Jones 1980). Monoxenic cultures of Me 10 idogyne spp. on plant roots have been used to study many aspects of the plant-pathogen biology such as juvenile migration inside the root (Nobre et al. 1995), giant 84
cellultrastructure (Stenderet al. 1986; Sijmonset al. 1991)and plant resistance (Huettel and Hammerschlag1986; BrownandMojtahedi 1989; Hashmi et al. 1994). Depending onthe hostplant andthe nematode species used, the studies employed various culture media, such as Murashige and Skoog medium for the infection of peach plantlets with Mel oidogyne incognita (Kofoid and White) Chitwood (Huette1 andHammerschlag 1986), Skoog, Tsuiand White mediumfor the infectionofexcised tomato roots with Meloidogyne javanica (Treub) Chitwood(Glazerand Orion 1984), and Gamborg's B5 medium for the infection of hairy root cultures from severalplants withM. javanica (Verdejo et al. 1988). Sijmons et al. (1991) conducted a systematic optimisation oftheculture conditions forthe infection of seedlings of Arabidopsis thaliana (L. Heynh.) with the beet cyst-nematodeHeterodera schachtii Schmidt. A modifiedKnop medium (Sijmons et al. 1991) was optimalfor infectionby H. schachtii, and also allowed infection of Arabidopsis by M incognita and Meloidogyne arenaria (Neat) Chitwood. In contrastto these results withArabidopsis, wefound Australasian Plant Pathology Vo!. 27 (2) 1998
that tomato seedlings grew poorly and supported low infection rates on modified Knop medium. Thereforewe optimisedcultureconditions forinfection of tomato seedlings by M javanica.
Methods Preparation of inoculum Nematode eggsusedin this study were obtainedfrom stock cultures maintained on glasshouse-grown tomato plants (Lycopersicon esculentum Miller cv. GrosseLisse). Twenty non-sterile egg masses were collected in a 1.5 mL polypropylene reaction tube containing 600 J.LL of steriledistilledwater. Theseweresurfacesterilisedin a laminarflowcabinetbyadding600 J!L of 5% NaOCl (final concentration2.5%, Le. 1.25% available chlorine) and shaking vigorously for 4 min. The eggsuspension was centrifuged at 1000g for 5 min and the supernatantremoved witha micropipette. Then, 1% (wlv)HgCl2 was added to a final concentration of 0.2% and the suspension was shaken gently for 4 min. The suspension was centrifugedat 1000g for5 min,the supernatant removed and the eggs were resuspended in 200 J.LL of sterile distilled water. The suspensionwas transferredinto a 10 mL capped conical centrifuge tube. The eggs were washed twice with 10 mL of sterile distilled water by incubating for 5 min and subsequentcentrifugation. After resuspension in 1 mL of sterile water, the eggs were ready for inoculation. Todetermine the concentrationof the inoculum, ten aliquots of 10 J.LL were collected while gently swirling the egg suspensionand individualdroplets were pipetted onto a 3-
