Acta vet. scand. 1989, 30, 77-87.

Induction of -Trapping Organs in the Predacious Fungus Arthrobotrys oligospora (Hyphomycetales) by Infective Larvae of ostertagi ()

By JfiJrn GrfiJnvold

Institute of Hygiene and Microb10logy, Royal Veterinary and Agncultural Umvemty, Denmark.

Grenvold, J.: Induction of nematode-trapping organs in the predacious fungus Ar• throbotrys o/igospora (Hyphomycetales) by infective larvae of Ostertag1a ostertag1 (Tnchostrongy/idae). Acta vet. scand. 1989, 30, 77-87. - Laboratory expenments were designed to study the mfluence of temperature, concentrations of , oxygen tension, light, and nutnent levels, on the mduction of nematode-trappmg hyphal nets in the predacious fungus Arthrobotrys o/igospora When induced by infective Ostertagza ostertag1 larvae, a maximum number of nets was produced at 20°C, at which temperature nets in surplus were produced at larval concentrations up to 1,000 larvae per cm2. A obgospora did not produce nets in an anaerobic atmosphere containing 21 % C02 (v/v), and net induction was suppressed to acer• tain degree by exposure to light. The composition of the medium had an important influence on the saprophytic growth and the net-forming capability of A o/igo• spora as a maximum number of nets was induced at a relatively low concentration of com meal supporting the relatively sparse mycehum. It was shown that a pro• portion of trapping nets in A. o/igospora maintained their trapping potential for more than 7 weeks when the temperature was below 25°C. Induction of nematode• trappmg organs m A o/igospora is discussed in relation to control of infective nematode parasite larvae in cow pats.

nematodes; parasites.

Introduction may be formed by hyphal anastomoses, and The predacious fungus Arthrobotrys oligo• eventually a three-dimensional network is spora is able to trap migrating nematodes in created (Fig. l ). three-dimensional adhesive nets raised abo• In A. o/igospora trapping nets are formed ve the surface of the medium. Net formation only if the fungus is stimulated (induced) by starts by lateral branch growing out from the factors of physio-chemical (e.g. electrical po• vegetative hyphae. This branch curls back tentials or peptides) or biological (e.g. nema• and anastomoses with the parent or adjecent todes and several other soil ) nature hyphae. From this primary loop other loops Pramer & Stoll (1959) were able to induce

Acta vet scand vol 30 no I - 1989

Induction ofnematode-trapping organs 79

1980). This is in accordance with results of body of the P. redivivus was filled with Ba/an et al. (1974) who found that the pro• hyphae (Nordbring-Hertz & Stdlhammer• duction of attractants in A. olzgospora was Carlemalm 1978). potentiated by traps formed in the presence Nansen et al. (1986) compared the preda• of nematodes. cious activity of A. oligospora on the pre• The nets in A. olzgospora are composed of parasitic stages of the cattle nematode para• hyphae that are more robust and with a cell site oncophora and the free-living wall often 4-5 times thicker than the vege• soil nematode P redivivus. The first and tative hyphae (Nordbring-Hertz & Stdlham• second larval stages of C. oncophora and mer-Carlemalm 1978). The trapping nets P. redivzvus were killed rapidly; in contrast, are covered with a mucilagineous coat that third-stage parasite larvae wriggled in the sticks to living nematodes, and there is ex• traps for many hours. This may be because perimental evidence for the presence of a it is time-consuming for A. oligospora to lectin on the net surface which binds to a penetrate the outer cuticle of the previous carbohydrate on the nematode cuticle moult which protects both C. oncophora and (Nordbrmg-Hertz & Mattiasson 1979, Mat• 0 ostertagi larvae in their third and infec• tzasson et al. 1980). The adhesion mecha• tive stage until infection of the host takes nism has been shown to be equally effective place. against a range of bacteria-feeding and plant• Generally, morphogenic processes are highly parasitic nematodes (Jansson & Nordbring• influenced by environmental factors. The se• Hertz 1980) as well as a range of -pa• ries of experiments presented in this paper rasitic nematodes (Nansen et al. 1988). Ob• was performed to investigate the influences servations by N ordbrmg-Hertz & Stdlham• of temperature, concentrations of nemato• mer-Carlemalm (1978) indicate that the des, oxygen tensions, light, and nutrient struggling of captured nematodes trigger an levels, on net formation in A. olzgospora in• increased secretion of adhesive substances duced by infective larvae of the cattle nema• between the fungal traps and the nematodes. tode parasite 0. ostertagi. The trapping ca• Penetration of trapped P. redivivus was ob• pability of nets of increasing age was invest• served within l h by Nordbrmg-Hertz & igated using the free-living nematode P. Stdlhammer-Carlemalm (1978). The me• redivivus. chanism by which the cuticle is penetrated is not known, but it is possible that it is partly mechanic and partly enzymatic (Barron 1981 ). Within 45 min. of penetration, an in• fective bulb is formed inside the trapped ne• Materials and methods matode (Shepherd 1955), at which time mo• General experimental conditions vements of the nematode ceased completely. The fungi were grown on diluted Com Meal Apparently nets in A. olzgospora secrete a Agar (Difeo) (CMA l: IO) (Lysek & Nord• nematotoxin which paralyses or kills trap• brmg-Hertz 1981): Temperature = 20"C; ped nematodes (Olthof & Estey 1963). From relative humidity = 90--100 %; pH = 6,5; this bulb, which reaches a size about half the normal atmosphere (21 % 0 2 (v/v)); and diameter of the nematode, trophic hyphae darkness. Only deviations from these con• grow out and extend throughout the body of ditions are mentioned in the following de• the nematode (Fig. l ). After 6 h the whole scriptions of single experiments.

Acta vet scand vol 30 no I - 1989 80 J. Grenvold

Saprophytic growth rate of different species each area. The average number of nets per ofArthrobotrys cm2 and the average number of loops per net This experiment was performed in large Pe• could then be calculated for each treatment. tri dishes (diam.: 8.5 cm). Five Arthrobotrys species (A. oligospora (A TCC 24927), A. musiformis, A. tortor (4-182-3-INRA), Net induction at different temperatu• A. conoides (CBS 109.52), and A. arthrobo• res. Cultures of A. oligospora were induced tryoides (CBS 118.54)) were inoculated in by 50 infective 0. ostertag1 larvae per cm2 at the centre of the dishes. Three Petri dishes each of the following constant temperatures: with each species were placed at each of the s·, 10·, is·, 20·, 25°, 30°, and 35°C. Three following constant temperatures: 5°, 1o·, 15°, petri dishes were incubated at each tempera• 20°, 25°, 30°, and 35°C. The radii of the ture. The numbers of nets and loops were colonies were measured after 3 days, and the counted after 3 days of contact with the pa• average saprophytic growth rate of each fun• rasite larvae. gal species at each temperature was calcul• ated in mm/3 days. Net induction at different concentra• tions of parasite larvae. Cultures of A. oligospora, grown on CMA I: I 0 contain• The influence of different factors on the ing 0.02 % tetracycling chloride, were indu• induction of trapping nets in Arthrobotrys ced by 10, 100, 300, 600, and 1,000 infec• oligospora tive 0. ostertagi larvae per cm2• The super• These experimentswere performed in small natant from the last larval washing was used Petri dishes (diam.: 3.5 cm; area: 9.6 cm2). in control Petri dishes without larvae. Tetra• The predacious fungus A. oligospora (A TCC cycline chloride was added to the medium to 24927) was inoculated in the centre of the suppress bacterial growth, especially in dis• Petri dishes. One week later, when the fun• hes with high larval concentrations, as the gus covered the whole surface, nets were in• larvae were non-sterile. For each larval con• duced by adding infective 0. ostertagi larvae centration, 2 Petri dishes were incubated at in 3 drops of non-sterile water to each cul• 20·c for 5 days, after which time nets and ture. Infective 0. ostertagi larvae had been loops were counted. kept in shallow water at S°C for I week to 3 months. Immediately before use, the larvae Net induction at different oxygen ten• were washed 3 times in tap water, using sions (v/v). Cultures of A. oligospora were centrifugation followed by decantation of the induced by 50 infective 0. ostertagi larvae supernatant. All larvae were living and mo• per cm2• Three Petri dishes were placed in tile when added to the fungal cultures. each of three 2.5-litre containers. In the first After a period of some days, the number of container 0 2 was replaced by C02 by Ana• trapping nets and the number of loops per erocultiBl A (Merck), in the second there was net were determined. The number of nets a normal atmosphere (21 % 0 2), and in the were counted on 5 randomly selected areas third there was a pure 0 2 atmosphere. The of 3. l 4 mm2 on the surface of each Petri pressure was 750-770 mm Hg. The contai• dish situated between the centre and the rim ners were incubated at 2o·c for 2 days, after of the dishes. The number of loops per net which time the numbers of nets and loops was counted on 2 randomly selected nets in per net were countered. During the follow-

Acta vet scand vol 30 no I - 1989 Induction of nematode-trapping organs 81 ing days all categories of Petri dishes were a number of these cultures at the following observed at normal atmosphere (2 l % 0 2). temperatures: 5°, 10·, 15°, 20·, 25°, 30·, and 35°C. At intervals (l, 2, 4, and 7 weeks after Net induction in light and darkness. the start of the experiment) 3 Petri dishes Cultures of A. oligospora were induced by from each temperature were exposed to a 50 infective 0. ostertagi larvae per cm2, and known number (between 40 and 70 indivi• incubated at 2o·c. Six Petri dishes were duals) of P. redivivus per cm2 for 2 h. No placed under constant electric light at 500 new trapping nets were produced during this Lux, while 6 other dishes were incubated in period. Not-trapped nematodes were isola• the dark. The trial was finished 7 days later ted from the dishes by Baermannization for by counting nets and loops per net. 16 h according to Jorgensen & Madsen (1982). Control Petri dishes without A. oli• Net induction at different concentra• gospora were treated in the same way. The tions of corn meal in the medium. The activity of the nets was calculated as the predacious fungus A. oligospora was culti• mean percentage of trapped P. redivivus in vated on agar containing an increasing Petri dishes inoculated with the fungus, m amount of com meal per litre (0.0, 0.1, 0.2, proportion to those in control dishes. 0.4, 1.0, and 2.0 g com meal/I). The me• dium was a mixture of Bacto Agar (Difeo) and Com Meal Agar (Difeo) with a constant amount of 15 g agar per litre (I. 5 %) at each medium concentration. One week old A. oli• Results gospora cultures were induced by 50 infec• Saprophytic growth rate of different species tive 0. ostertagz larvae per cm2• Three cul• ofArthrobotrys tures of each category were incubated at As shown in Table I, the tested Arthrobotrys 20-C for 3 days, after which time the num• species had growth rate optima at about 20· bers of nets and loops per net were deter• or 25°C. The fungi A. oligospora, A. musi• mined. formzs, and A. tortor are fast-growing spe• cies. A. oligospora and A tortor grow well in The influence of age and temperature on the the interval from 15° to 30°C. A. muszformis actzvzty of nets zn Arthrobotrys olzgospora grows well between 15° and 25-C, but no This experiment was performed in small Pe• growth was observed at 30°C. All species, tri dishes (diam.: 3.5 cm; area: 9.6 cm2) on except A. olzgospora, grew slowly at S°C, but CMA 1:10 containing 0.02 % tetracycline none of the species showed any activity at chloride. Tetracycline chloride was added to 35·c. the medium to suppress bacterial growth during the long experimental period of 7 The influence of dzjferent factors on the weeks. One week old cultures of A. oligo• induction of trapping nets in Arthrobotrys spora were induced by 60 non-sterile indivi• oligospora duals of P. redivivus per cm2 at 2o·c for 2 Net induction at different temperatu• days. At that time, nets in excess were pre• res. Temperature had a significant influence sent in each culture (approx. 600 nets per on the production of trapping nets in A. oli• cm2). gospora when the fungus was induced by 50 The experiment started on day 2 by placing infective 0. ostertagi larvae per cm2 (Fig. 2).

Acta vet scand vol 30 no I - 1989

Inductwn of nematode-trapping organs 85 periment. On the other hand light, whether multiply, the only result of predacious acti• continuous, intermittent, or absent, has little vity is a reduction of the population of infec• influence on the saprophytic growth rate tive larvae. Furthermore, old nets may act as (Olthof & Estey 1965). survival structures. The composit10n of the medium has an im• portant influence on the saprophytic growth and the net-forming capability in A. olzgo• Acknowledgements spora. Nematodes alone cannot provide the The author wishes to express his gratitude to Dr. sole energy for A. oligospora (Hayes & Blfg1t Nordbnng-Hertz, Lund umvers1ty, Sweden, and to Dr. Michele Peloille, INRA, Monna1e, Blackburn 1966). In their work, Hayes & France, for prov1dmg the fungal cultures. Blackburn (1966) showed that the vitamins Ms. Annette Komer 1s thanked for valuable tech• acid, biotine, thiamine, and p-aminobenzoic mcal assistance. The author is also indebted to stimulated saprophytic growth but had little Ms. Jane R0ken for correcting and typing the effect on predacity. Nitrogen supplied as ni• manuscnpt. trate, nitrite, or urea supported both growth The work was supported by grant No. 13-3716 and predacity in A digospora. Hayes & from the Damsh Vetennary and Agncultural Re• Blackburn (1966) also found that a range of search Council. carbohydrates was able to support saprophy• tic growth in A. olzgospora. However, exten• References sive mycelial growth was not always corre• Ba/an J, Krzzkofo L, Nemec P, Vollek V Pro• lated with high predacity. On some carbo• duct10n of nematode-attractmg and nemat1c1- hydrates A oligospora produced little myce• dal substances by predacious fung1. Folia Mi• lium, but predacity was high. Nordbnng• crob101. 1974, 19, 512-519. Hertz (1973) found that on low-nutrient me• Barron GL Predators and parasites of m1crosco• dia phenylalanyl-valine induced trap forma• p1c ammals. In: Biology of Comd1al Fungi tion in A olzgospora, but the effect was (Cole GT, Kendnck K, eds.). Academic Press, greatly reduced or annulled in a nutrient• New York 1981,2, 167-200. RC Ecological charactenstics of nema• rich medium or on water agar. These infor• Cooke tode-trappmg Hyphomycetes I. Preliminary mations correspond with the present results studies. Ann. appl. Biol. 1963, 52, 431-437. where maximum numbers of nets were indu• Cooke RC Ecolog1cal charactenstics of nema• ced at a relatively low concentrat10n of corn tode-trappmg Hyphomycetes IL Germmat10n meal which supported a relatively sparse of comdia in soil. Ann. appl. Biol. 1964, 54, mycelium. Based on the above-mentioned 375-379. results it is possible that the nutrient level in Eren J, Pramer D Growth and activity of the cow pats are not always optimal for net nematode-trappmg fungus Arthrobotrys cono- induction. 1des m soil. In: Microbial Ecology (Loutit The long survival of trapping nets at high MW, Miles JAR, eds.). Springer-Verlag, Ber• lm 1978, 121-127. humidity and temperatures below 25°C is Gronvold J, Korsholm H, Wolstrup J, Nansen P, surprising, as vegetative hyphae were ob• Henriksen SvAa Laboratory experiments to served to collapse within a couple of weeks. evaluate the ability of Arthrobotrys ohgospora The present results indicate that capture of to destroy mfect1ve larvae of Cooperza species, infective parasite larvae in the cow pats may and to mvest1gate the effect of physical factors continue for a long time under suitable con• on the growth of the fungus. J. Helmmthol. ditions. As parasite nematode larvae do not 1985, 59, 119-125.

Acta vet scand vol 30 no I - 1989 86 J. Grenvold

Grenvold J, Nansen P, Henriksen SvAa, Thylm J, Nordbrmg-Hertz B. Nematode-induced morpho• Wolstrup J· The capabihty of the predacious genesis in the predacious fungus Arthrobotrys fungus Arthrobotrys o/zgospora (Hyphomyce• olzgospora. Nematologica 1977, 23, 443-451. tales) to reduce numbers of infective larvae of Nordbrzng-Hertz B, Brink C Qualitative charac• Ostertagia ostertagz (Trzchostrongylzdae) in terization of some peptides inducing morpho• cow pats and herbage during the grazing sea• genesis in the nematode-trapping fungus Ar• son in Denmark. J. Helminthol. 1988, 62, throbotrys o/zgospora Physiol. Plant. 1974, 271-280. 31, 59-63. Hayes WA, Blackburn F Studies on the nutri• Nordbrzng-Hertz B, Mattiasson B· Action ofa ne• tion of Arthrobotrys olzgospora Fres. and A matode-trapping fungus shows lectin-media• robusta Dudd. II. The predaceous phase. Ann. ted host-microorganism interaction. Nature appl. Biol. 1966, 58, 51-60. 1979,281,477-479. Jansson H-B, Nordbrmg-Hertz B Interactions Nordbrmg-Hertz B, Odham G Determination of between nematophageous fungi and plant-pa• volatile nematode exudates and their effects rasitic nematodes: Attraction, induction of on a nematode-trapping fungus. Microb. Ecol. trap formation and capture. Nematologica. 1980, 6, 241-251. 1980,26, 383-389. Nordbrzng-Hertz B, Stdlhammer-Carlemalm M: Jergensen RJ, Madsen K. Unders0gelse af freces• Capture of nematodes by Arthrobotrys olzgo• pr0Ver for lungeormlarver. (Examination of spora, an electron microscope study. Canad. faecal samples for lungworm larvae). Dansk J. Bot. 1978, 56, 1297-1307. VetTidsskr. 1982, 65, 517-518. Olthof HA, Estey RH A nematotoxin produced Lysek G, Nordbrzng-Hertz B An endogenous by the nematophagus fungus Arthrobotrys olz• rhythm of trap formation in the nematopha• gospora Fresenius. Nature 1963, 196, 514- gous fungus Arthrobotrys olzgospora. Planta. 515. 1981, 152, 50-53. Olthof HA, Estey RH. Relation of environmental Mankau R. Soil fungistasis and nematophagus factors to growth of several nematophagous fungi. Phytopathology 1962, 52, 611-615. Hyphomycetes Canad. J. Microb. 1965, 11, Mattzasson B, Johansson P-A, Nordbrzng-Hertz 939-946. B: Host-microorganism interaction; Studies on Pramer D, Stoll NR Nemin: a morphogenic sub• the molecular mechanisms behind the capture stance causing trap formation by predaceous of nematodes by nematophagous fungi. Acta fungi. Science 1959, 129, 966-967. Chem. Scand. 1980, 34, 539-540. Rose JH. Some observations on the free-living Nansen P, Grenvold J, Henriksen SvAa, Wolstrup stages of Ostertagza ostertagz, a stomach worm J: Predacious activity of the nematode-de• of cattle. Parasitology 1961, 51, 295-307. stroying fungus, Arthrobotrys oligospora, on Shepherd AM Formation of the infection bulb preparasitic larvae of and in Arthrobotrys olzgospora Fresenius. Nature on soil nematodes. Proc. Helminthol. Soc. 1955, 175, 475. \Vash. 1986,53, 237-243. Nansen P, Grenvold J, Henriksen SvAa, Wolstrup Sammendrag J: Interactions between the predacious fungus Induktwn of nematod-fangende organer hos rov• Arthrobotrys o/zgospora and third-stage larvae svampen Arthrobotrys olzgospora (Hyphomyceta• of a series of animal-parasitic nematodes. Vet. les) ved h1alp af znfektzve larver af Ostertagia parasitol. 1988, 26, 329-337. ostertag1 (Trzchostrongylzdae) Nordbrmg-Hertz B: Peptide-induced morphoge• Ved en serie laboratorie-fors0g blev indflydelsen nesis in the nematode-trapping fungus Arthro• af temperatur, koncentration af nematoder, ilt• botrys olzgospora Physiol. Plant. 1973, 29, tension, lys og substratstyrke malt som antallet af 223-233. nematod-fangende svampe-net, der kunne induce• res hos rovsvampen Arthrobotrys oligospora. Nar

Acta vet scand vol 30 no I - 1989 Induction of nematode-trapping organs 87 rovsvampen blev induceret med infektive Osterta• gerende indflydelse pa vreksten og net-dannelsen gia ostertag1 Jarver fandtes en maksimal produk• hos A oligospora, idet en maksimal produktion af tion af net ved 2o·c. ved denne temperatur dan• net kunne induceres ved en relativ !av koncentra• nede rovsvampen net i overskud ved koncentratio• tion af maj me!, som gav anlednmg ti! et relativt ner aflarver op ti! 1.000 Jarver pr. cm2• A. oligo• sparsomt mycelium. En de! af nettene hos A oli• spora dannede ikke net under anaerobe for• gospora bevarede deres fangst-potentiale i mere hold i en atmosfrere indeholdende 21 % C02 end 7 uger ved temperaturer lavere end 25°C. In• (rumfang/Rumfang), og induktionen af net blev duktion af nematod-fangende organer A oligo• undertrykt ti! en vis grad, mi.r rovsvampen var ud• spora d1skuteres i relation ti! kontrol af infektive sat for lys. Mediets sammensretmng havde en af- paras1tiske nematodlarver i kokasser.

(Received May 20, 1988, accepted June 29, 1988)

Reprints may be requested from: J0m Gnmvold, Institute of Hygiene and Microbiology, Royal Vetennary and Agricultural University, 13, Biilowsvej, DK-1870 Frederiksberg C, Denmark.

Acta vet scand vol 30 no I - 1989