C.Ü. Fen-Edebiyat Fakültesi
Fen Bilimleri Dergisi (2008)Cilt 29 Say 1
Gnotobiotic Culture of Zygotylenchus guevarai (Nematoda: Pratylenchidae) on Carrot Discs
Mehmet KARAKA
Ankara University, Science and Engineering Faculty, Department of Biology 06100 Tando an-Ankara, TURKEY
e-mail: mkarakas@science.ankara.edu.tr
Received: 30.04.2007, Accepted: 24.01.2008
Abstract: Zygotylenchus guevarai (Nematoda: Pratylenchidae) obtained from a field population was cultured on parsley at 25 C and 70% relative humidity in a growth chamber. Nematodes were surface sterilized using solutions of streptomycin sulfate (SS) and either mercuric chloride (HgCl2). Effectiveness
of the disinfectants was evaluated by plating surface sterilized nematodes on potato-dextrose agar and by incubating in beef nutrient broth for 7 days at 23 C. Zygotylenchus guevarai were axenic and remained viable after soaking in a disinfectant containing 100 ppm HgCl2 and 1000 ppm SS for 2 minutes followed
by two rinses with sterile distilled water. Nematodes were surface sterilized following migration through agar containing 5 ppm HgCl2 and 10 ppm SS. Viable axenic nematodes obtained after soaking and
migration in HgCl2 and SS were injected into fresh carrot discs and incubated at 23 C. At 64 days,
population increases resulting from these axenizing techniques were not significantly different (P = 0.10). Key Words: Zygotylenchus guevarai, root lesion nematode, gnotobiotic culture
Havuç Disklerinde Zygotylenchus guevarai (Nematoda: Pratylenchidae)’nin Gnotobiyotik Kültürü
streptomisin sülfat (SS) ve civa klorür (HgCl2) solüsyonlar kullan larak, yüzey sterilizasyonlar
sa lanm r. Dezenfektanlar n etkisi, yüzey sterilizasyonu sa lanm nematodlar n patates-dekstroz agardaki geli imleri ve et sulu besin ortam nda 23 C de 7 gün içindeki ço almalar na göre de erlendirilmi tir. Zygotylenchus guevarai’nin, 100 ppm HgCl2 ve 1000 ppm SS içeren dezenfektanda 2
dakika tutulmas n ard ndan, iki defa steril saf sudan geçirildikten sonra canl olarak dekontaminasyonu sa lanm r. Yüzey sterilizasyonunun sa lanmas n ard ndan nematodlar, 5 ppm HgCl2 ve 10 ppm SS
içeren agar ortam nda yay göstermi lerdir. HgCl2 ve SS uygulamalar ndan sonra canl koruyan
dekontamine nematodlar, taze havuç disklerine a lanm ve 23 C de üretilmi lerdir. Bu dekontaminasyon tekniklerinden, 64 günde saptanan populasyon art ndaki sonuçlar önemli fark göstermemi tir (P = 0.10).
Anahtar Kelimeler: Zygotylenchus guevarai, kök lezyon nematodu, gnotobiyotik kültür
INTRODUCTION
Nematode culture on potted host plants requires daily maintenance of the host and typically provides a nematode source contaminated with numerous soil inhabiting microorganisms. Culturing the root lesion nematode, Zygotylenchus guevarai (Tobar Jiménez, 1963) Braun and Loof, 1966 at the optimum reproductive temperature of 25 °C [1] requires the use of a growth chamber or temperature tank.
In earlier reports of gnotobiotic nematode culture (-with known associated organisms or none), disinfectants such as Aretan that contained a low percentage of organic mercury were effective, but are no longer easily obtainable. Technical grade Ceresan Nass, a methoxyethylmercury chloride compound, and HgCl2 are potentially more toxic than Aretan. Surface sterilization of Aphelenchoides ritzemabosi
(Schwartz, 1911) Steiner and Buhrer, 1932 and Radopholus similis (Cobb, 1893) Thorne, 1949. with various concentrations of mercuric chloride and several rinses of sterile distilled water have been reported [2,3]. The agar migration method with Aretan was successful with Paratrichodorus christiei (Allen, 1957) Siddiqi, 1974 and
Pratylenchus vulnus Allen and Jensen, 1951. Since Ceresan Nass and HgCl2 are
available, evaluation of their toxicity and efficacy for surface sterilization of previously untested nematode species was needed. In this study I described two methods for producing axenic (-no associated organisms) carrot disc cultures of Z. guevarai using HgCl2 and SS as surface sterilants.
MATERIALS AND METHODS
Z. guevarai obtained from infested parsley roots in K lcahamam, Turkey (40°
28.8' N 32° 39.0' E) and was cultured on parsley seedlings in sand-loam soil in a growth chamber at 25 °C and %70 relative humidity. Following mist chamber extraction [4] individual Z. guevarai were removed from the nematode suspension using a nylon nematode pick. This monospesific population was reared on carrot discs for two months and then used as a nematode source for the two axenizing techniques studied.
Agar migration: Approximately 50,000 Z. guevarai were concentrated into 2 ml
of water and added to 70 ml of %1 water agar cooled to 40-45 °C. The nematode agar suspension was distributed evenly into 10 sterile petri dishes. Concentrations of 5 ppm HgCl2 was mixed with 10 ppm SS and added to additional %1 water agar cooled to 45 °C [7]. The amended water agar was then poured onto the solidified nematode agar suspension to a depth of 5 mm in each petri dish. These were incubated at 23 °C for 36 hours. Nematodes which had migrated to the agar surface were rinsed off the agar with 10 ml of sterile distilled water, counted, and concentrated into 0.5 ml prior to injection into carrot discs. Twelve replicates of 10 petri dishes each were conducted.
Soaking: One hundred Z. guevarai were concentrated in 0.5 ml of water in a test
tube to which 5 ml of disinfectant containing 100 ppm HgCl2 and 1000 ppm SS was added [7]. The contents were agitated, allowed to settle for 2 minutes and centrifuged at 1000 relative centrifugal force for 45 seconds. The disinfectant was decanted immediately to a 0.5 ml volume and the nematodes were rinsed twice by centrifuging aseptically with 5 ml of sterile distilled water. The supernatant was decanted so that a 0.5 ml volume remained for injection into the carrot discs. The soaking protocol was repeated twelve times.
When determining the mortality rate of Z. guevarai using these axenizing techniques, I assumed that a dead nematode or one unable to penetrate carrot tissue would lay outstretched or in an open “c” form with no movement [5,6]. Effectiveness of these axenizing techniques was determined by incubating the treated nematodes on potato dextrose agar (PDA) and in beef nutrient broth (BNB) for 7 days at 23 °C.
Preparing carrot discs for nematode culture: Carrots placed in lukewarm soapy
a clean tub and added carrots. Ascertain that carrots were completely submerged in bleach solution. Carrots left in solution for 30 minutes and drained.
Carrots placed on paper towels on trays and covered with paper towels and transferred on tray to a laminar airflow chamber. Remaining operations are to be done in chamber. In the airflow chamber, sterilized a knife by dipping it in %95 alcohol and passing it over a flame. Cut off the crown end of the carrot. Hold carrot at the tapered end and using an alcohol dipped and flamed vegetable peeler. Carrot peeled downwards in slightly overlapping longitudinal strips. Peeler sterilized after every two strips. Peeled portion should be of a diameter small enough to fit into a wide mouth flint glass bottle.
Using an alcohol dipped and flamed sharp knife, the pealed carrot portion cut approximately 30 to 40 mm in length and not less than 20 mm wide cylindrical pieces. The knife sterilized after every cut. Cut carrot cylinders transferred to autoclaved wide mouth flint glass bottles or jars using a set of sterilized forceps. The forceps sterilized after every cylinder. Per bottle put at least four cylinders.
Labeled bottle with tightened bottle cap stored in dark at 24-25 °C. After 1-2 weeks, or as soon as white specks of callus were visible on the carrots, inoculated with nematodes.
Carrot disc culture: Approximately 100 surface sterilized Z. guevarai were
injected under carrot tissue with a sterile 20 gauge syringe. Nematodes did not need to be surface sterilized but should be rinsed in a few changes of sterile water and inoculated in a few milliliters of the same. Either four or five nematode-inoculated discs were maintained in previously autoclaved 150 ml fleakers incubated at 23 °C. Population increases were determined after 64 days. Nematodes were extracted by slicing the carrot discs into 5 mm thick cross sections and incubating the sections in a mist chamber at 18 °C for 16 days. Mean population differences between the two axenizing techniques were compared using one-way ANOVA analysis.
RESULTS AND DISCUSSION
Z. guevarai survived in the disinfectant containing 100 ppm HgCl2 and 1000 ppm SS for up to 120 minutes, however, after 30 minutes nematode activity decreased. What effects this apparent toxicity had on the nematode infectivity and reproductive capacity could not be determined in this study. Treatment of Z. guevarai for a minimum
of 2 minutes in a 100 ppm HgCl2 plus and 1000 ppm SS solution followed by two rinses with sterile distilled water was sufficient to inhibit growth of secondary microorganisms when plated on PDA or incubated in BNB.
The average number of nematodes recovered after migration through agar containing HgCl2 varied from 50 to 180 per trial (Table 1). This represent only those active individuals because it was assumed that nematode death could have occurred following their upward migration to the agar surface. No microorganisms were associated with these nematodes on PDA or in BNB. Variability in numbers of living nematodes recovered from the 12 trials using this technique may have resulted from slight variations in depth of agar among petri dishes through which the nematodes migrated. Nematode migration might be enhanced by incubating the plates at temperatures greater than 23 °C.
Numbers of Z. guevarai recovered and their population increases were not different (P = 0.10) when comparing the two HgCl2 and SS axenizing techniques (Table 1). The low water temperature in the mist chamber probably restricted nematode migration from the discs and more nematodes would have been extracted in less time if the water temperature had been higher. Greater increases also would be expected if discs were incubated at 25 °C which is the optimum temperature for Z. guevarai. Large numbers of second-stage juveniles began to emerge after 7 days as eggs hatched, but were not counted in the population.
Following nematode inoculation, healthy carrot discs were characterized by white callus growth [7,8,9,10]. Sandstedt and Schuster [11] observed greater callus growth at the radical end of carrot discs when inoculated with Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 and suggested that this was partly due to enhanced nematode nutrition at the radical end. A similar growth pattern of callus tissue was observed in this study, but discs were placed randomly in the fleakers resulting in both apical and radical ends of discs being inoculated, which may explain partly why nematode increases varied between replications.
Table 1. Populations of Zygotylenchus guevarai recovered following treatment with disinfectants containing mercuric chloride (HgCl2) and streptomycin sulfate (SS) and incubation in carrot discs for 64
days.
Axenizing technique Number inoculated Number recovered* (x 1000) Population increase* (x 100) Agar migration 52 26 5 70 62 9 68 154 23 96 158 16 182 322 18 90 110 12 5 ppm HgCl2 + 10 ppm SS 72 96 13 94 131 14 98 146 15 80 134 17 85 120 14 74 114 15 2 minutes soak 100 90 9 100 75 8 100 136 14 100 55 6 100 140 14 100 190 19 100 ppm HgCl2 + 1000 ppm SS 100 184 18 100 198 20 100 280 28 100 160 16 100 274 27 100 112 11
*No significant differences between axenizing techniques in either number recovered or population increase
Variability in the life stages of the initial nematode inoculum also may account for the differences. With time, the white callus growth changed to a brown-bronze color[12]. Some carrot tissue began degrading after 64 days. The carrots used for
establishing these cultures may have had internal bacterial contamination. I have maintained other cultures more than 70 days at 25 °C and have observed no contamination, indicating that a temperature relationship may occur[13,14,15,16].
Nematodes were observed migrating from discs as they moved up the jar walls. An efficient method of transferring nematodes to fresh carrot discs was to cut a 5 mm slice from a healthy callus covered disc, quarter the disc, and place it a top fresh discs for 5 days. Within 4 or 5 days a sufficient number of Z. guevarai was transferred with minimal contamination.
I have maintained monoxenic cultures (-with one other known associated organism) of Z. guevarai for ten mounts. The techniques developed and described in this paper have provided large numbers of axenic nematodes for other studies and should provide researchers studying other plant parasitic nematodes such as root lesion nematodes a method for producing monoxenic cultures.
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