1. Introduction

Species of the Dactylaria-complex are able to capture vermiform nematodes with trapping devices (Stirling, 1991). The infection process generally can be distinguished in different stages: attraction and capture, penetration, subsequent toxification/ inactivation and digestion of the nematodes by the fungus.

Most in vitro studies on the successive events of the infection process were performed on a model system with Panagrellus redivivus, a bacteriophagous nematode, and an isolate of the fungus Arthrobotrys oligospora (Tunlid et al., 1972). On capture and infection of plant-parasitic nematodes few reports are presented and little is known about the physiological and biochemical processes of the fungus-nematode interaction (Den Belder, 1991).

Several nematode-capturing fungi seem to be highly specific in attraction, capture and infection of particular nematode species (Jansson & Nordbring-Hertz; 1980; Boag et al., 1988; Esser et al., 1991). Other parasites of nematodes such as the bacterium Pasteuria penetrans have shown specificity in attachment to different populations of the same nematode species e.g. Meloidogyne spp. Thus results obtained with other than plant-parasitic nematodes may have a limited value in predicting capture ability of the latter.

Depending on their relative saprophytic/predacious abilities nematophagous fungi have been divided into three ecological groups (Jansson and Nordbring-Hertz, 1980). Group 1 , considered as the most saprophytic, constitutes the adhesive network formers. The nematode trapping rings structures are formed as a result of contact with nematodes. The fungi show good saprophytic growth on artificial media and are only weakly predatory.

Group 2 constitutes fungi producing other types of traps: (non-)constricting rings, adhesive knobs and adhesive branches. Trap formation is usually spontaneous in the absence of nematodes. These fungi show a higher predacious ability and weaker saprophytic ability than the group of the adhesive network formers.

Group 3 constitutes the endoparasitic fungi, which do not form any mycelium in soil. They persist in the soil as conidia or exist as zoospores. Most of these fungi are obligate nematode parasites.

Monacorsporium cionopagum (CBS 228.52) is a constricting ring former, Arthrobotrys dactyloides (CBS 109.37) as well as Arthrobotrys conoides (CBS 265.83), Arthrobotrys oligospora (CBS 115.81 and ATCC 24927), Arthrobotrys scaphoides (CBS 226.52) and Duddingtonia flagrans (CBS 565.50) belong to the above-metioned group 1 and are adhesive network formers.

Microorganisms need to be active at prevailing soil conditions in the field and to survive abiotic conditions that may occur during the day, in order to be effective and widely applicable as biological control agents against soil-inhabiting nematodes. Gronvold (1989) found a significant effect of temperature on the adhesive network development in Arthrobotrys oligospora (ATCC 24927): mycelium did not respond to juveniles or responded only slowly with the development of networks at temperatures below 15 °C or above 25 °C. Also Dactylella spp. between 20 and 24 °C captured higher proportions of nematodes captured than at lower temperatures (Feder, 1963).

The nutrients available in the environment of the fungus have effects on its metabolism and its morphognesis (Esser et al., 1991). Variations of the nutrient source showed that development of adhesive networks is highly affected by nutrients available (Gronvold, 1989). For example, isolates of A. oligospora developed poorly networks on water agar whereas vegetative hyphae developed normally (Soprunov, 1966; Nordbring-Hertz, 1977; Jansson and Nordbring-Hertz, 1980).

Morphological responses to light has been described for many fungi (Leach, 1971). Gronvold (1989) reported that light suppresses development of ring structures in A. oligospora (ATCC 24927).

Another key factor which plays a role in trapping activity is the age of the hyphae. Loss of virulence of old cultures of nematode-capturing fungi was observed by Couch (1937) and Feder (1963). Such loss of virulence by nematode-capturing fungi is of special significance because it may limit their usefulness for nematode control. More recent work by Heintz (1978) showed that ageing of mycelium of A. dactyloides and A. cladodes resulted in a reduction of the ability to capture nematodes. Also loss of adhesiveness of ring structures of Dactylella megalospora was found within seven days (Esser et al., 1991). Reduction of capture in ring structures of A. oligospora (ATCC 24927) also occured, when fungal colonies were kept for seven weeks at temperatures between 5 and 35 °C (Gronvold, 1989).

Products based on trapping device forming strains of the fungus Arthrobotrys (e.g. Arthrobotrys superba and Arthrobotrys irregularis) have been described (FR-A-2 402 412 and Cayrol et al., 1991) but these strains show delayed action against infective nematodes in soil due to the time needed for induction and formation of trapping devices. This delay in action results in limited protection of the plants against direct attack of infective stages of nematodes in the early stages of plant growth. This effect of the nematicidal preparations based on trap-forming Arthrobotrys strains is noticed primarily in the lower reproduction rate of the nematodes and the resulting low population in the next years and not in direct protection of the plants.

It has been found surprisingly that the capture of nematode by fungi is not only realizable by complex capture structures, but can also be accomplished by not visibly differentiated vegetative hyphae.

A special strain of Arthrobotrys oligospora was identified which is able to capture nematodes with undifferentiated mycelium. This strain (CBS 289.82) combines the properties of the above mentioned group 1 and group 2 nematophagous fungi viz. good saprotrophic growth and the ability to capture nematodes without the need for induction and formation of trapping devices. This enables Arthrobotrys oligospora strain CBS 289.82 to capture the infective stages of plant-parasitic nematodes in soil without delay to due induction and formation of trapping structures. This treat distinguishes Arthrobotrys oligospora strain CBS 289.82 from other strains of Arthrobotrys used in nematicidal products. Arthrobotrys oligospora strain CBS 289.82 has novel qualities as nematicidal product because of its unique way of capturing nematodes and the resulting ability to effectively protect plants from direct attack by infective stages of plant-parasitic nematodes.

2. Description of the invention

The invention relates to the use of nematophagous fungi to control plant parasitic nematodes. The invention further relates to a process for protecting plants against the action of plant parasitic nematodes, which comprises treating the plants, parts of the plants or the soil with nematophagous fungi having undifferentiated hyphae which are able to capture infective stages of plant-parasitic nematodes in the soil. The term nematode used hereafter refers to the infective stages of plant parasitic nematodes. In an preferred embodiment the fungus Arthrobotrys oligospora deposited under the number 289.82 at the Centraalbureau voor Schimmelcultures, Baarn (CBS 289.82) is used.

More generally the invention relates to a process for obtaining those fungi.

The nematophagous fungi are selected on basis of capacity of the hyphae of the fungi to attach to the nematode not only with the ring-structures, but with the whole length of their mycelium. This selection can be performed for example as follows:

1. Growing the fungi on an appropriate medium or other substrates until a definite mycelial mat has been formed,

2. Pipetting the nematodes on the mycelial mat and allow them to remain for an sufficient time, e.g. for five to ten minutes on the mat,

3. Washing the fungal culture from the faucet, e.g. by by placing it directly in the jet of tap water,

4. Examining the fungal mycelium whether the nematode have attached only to the special trapping devices or also to the non differentiated hyphae.

The nematocidal fungus identified according to instant invnetion is normally applied in form of compositions together with one or more agriculturally acceptable carriers, and can be applied to the crop area or plant to be treated, simultaneously or in succession, with further compounds.

These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, other nematicides or mixtures of several of these preparations, if desired together with futher carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation.

Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

The number of applications and the rate of application depends in the intensity of infestation by the corresponding nematodes, the kind of soil, the temperature and the plant.

The active ingredients can also act via the soil by impreganting the locus of the plant with a liquid composition, or by applying the compounds in solid form to the soil, e.g. in granular form (soil application).

The microorganism can be formulated in known manner to powder, e. g. wettable or soluble powders, pelletts, granules, etc. using a conventional formulating machine, emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, dusts, granulates, and also encapsulations, for example, in polymer substances, preferably biopolymers.

The methods of application, such as spraying, atomizing, dusting, scattering, coating or pouring, are chosen in accordance with the intended objectives and the circumstances. Also according to the circumstances the composition will be applied within a range of 0,01 to 50 kg active ingredient (a.i.)/ha (about 2.471 acres). Advantageous rates are ranging from 0.05 to 5 kg/ha, preferably from 0.1 to 2 kg/ha, especially prefered from 0.2 to 1 kg/ha.

The formulation, compositions or preparations containing the active ingredients and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, for example by homogenously mixing and/or grinding the active ingredients with extenders, for example solvents, solid carriers and, where appropriate, surface- active compounds (surfactants). The surfactants customarily employed in the art of formulation are described, for example, in "McCutcheon's Detergents and Emulsifiers Annual," MC Publishing Corp. Ringwood, New Jersey, 1979, and Sisely and Wood, "Encyclopedia of Surface Active Agents", Chemical Publishing Co., Inc. New York, 1980.

The particles can be applied directly to the soil or the substrate or used to dress or coat the plants or parts of the plants, especially the seeds. This results in a protection of the seeds from the nematodes and automatically in improved germinating efficiencies.The composition according to instant application is therefore especially suitable to be applied as a coating of plant seeds by impregnating the seeds either with a liquid formulation containing active ingredients, or coating them with a solid formulation. A process to prepare a coated microbial pesticide of antagonistic microorganisms has been described for example in WO 92/20229.

The compositions usually contain from about 0.1 to about 99 %, preferably about 0.1 to about 95 %, and most preferably from about 3 to about 90 % of the active ingredient, from about 1 to about 99.9 %, preferably from about 1 to about 99 %, and most preferably from about 5 to about 95 % of a solid or liquid adjuvant or other additives, and from about 0 to about 25 %, preferably about 0.1 to about 25 %, and most preferably from about 0.1 to about 20 % of a surfactant.

In a preferred embodiment the invention relates to a biopesticide composition for controlling the growth of nematodes comprising an nematophagous fungus having virulence against the targeted nematode in association with an agricultural carrier selected from a liquid, powder, granule. The liquid may be preferably water, oil or a wetting agent. The fungus may be especially in the form of mycelia when the carrier is a liquid.

The term fungus as used herein encompassess all parts of the fungus as for example spores, resting spores, chlamydospores, conidia and the mycelium. The composition which may be used to treat the plants may be especially in the form of fungal conidiae or mycelium.

The fungal cells or conidiae are employed preferably within the range of about 10 ⁴ to 10 ¹⁴ /g. further prefered 10 ⁵ to 10 ⁹ /g, especially prefered 10 ⁵ to 10 ⁷ /g dried fungal composition or ml fungal suspension depending on the kind of formulation and the circumstances, e. g. the kind of plant to be treated or the kind of soil.

The composition can be used to control plant parasitic cyst, root-knot and lesion nematodes, especially root-knot nematodes. In an preferred embodiment the composition is used to control Meloidogyne hapla and Meloidogyne incognita belonging to the Meloidogynidae. This family is closed related to also the potato cyst nematode and the sugar beet cyst nematode. Other plant parasitic nematodes are for example Pratylenchus spp., Radopholus similis, Oitylenchus dipsaci, Tylenchulus semipenetrans, Heterodera spp., Aphelanchoides spp., Longidorus spp., Xiphinema spp. and Trichodorus spp.

The composition may also contain other fungi which act in the same way and which improve the nematicidal effect. The composition may also contain other suitable additives as for example sustaining, protective nutrient carriers.

These nutrient carriers include for example carbon and nitrogen sources, peptone, trace elements, vitamins. Any material which enhances the viability of the fungus is suitable. Sustaining substances, e. g. sugars, alcohols or glycerin, may inhibit undesired drying of the fungus. The composition may also contain substance which act antioxidativ, e.g. vitamin C.

Plants which can be protected may be either monocotyledons or dicotyledons. Examples of families that are of special interest are Solanaceae and Brassicaceae. Examples of species of commercial interest that can be protected include:

- tobacco, Nicotiana tabacum L. - tomato, Lycopersicon esculentum

- potato, Solanum tuberosum L, - petunia, Petunia hybrida (Solanaceae)

- Canola/Rapeseed, - Brassica napus L,

- cabbage, broccoli, kale etc., - Brassica oleracea L,

- mustards Brassica juncea L, - Brassica nigra L,

- Sinapis alba L (Brassicaceae),

- sugar beet, Beta vulgaris,(Chenopodiaceae),

- cucumber, Curcurbita sp. (Curcurbitaceae),

- cotton, Gossypium sp., (Malvaceae),

- sunflower, Helianthus annuus,

- lettuce Lactuca sativa, (Asteraceae=Compositae),

- pea, Pisum sativum,

- soybean, Glycine max and alfalfa, Medicago sp. (Fabaceae=Leguminoseae),

- asparagus, Asparagus officinalis; - gladiolus, Gladiolus sp., (Lilaceae);

- corn, Zea mays and - rice, Oryza sativa (Poaceae).

In an preferred embodiment the nematicide prevents diseases of plants such as potato, tomato, wheat, cabbage and Chinese cabbage.

It has been found that capture of nematodes by adhesive hyphae of Arthrobotrys oligospora (CBS 289.82) is very effective under a broad range of conditions which can occur in the soil, e.g. temperature, nutrient level and light. At temperatures occuring in the field, e. g. between 5 and 35 °C this fungal isolate and the juveniles of this nematode species are both sufficiently active to become firmly attached to each other, resulting in the arrest, killing and digestion of the nematodes.

The temperature-independent capture of nematodes by hyphae appears quite exceptional in comparison with fungi that capture nematodes through more complex structures only.

Hyphae of A. oligospora (CBS 289.82), developing on water agar and those growing on low nutrient salt media or corn meal agar, are able to capture all M. hapla juveniles present within one hour. Under nutritional conditions ranging from simple to more complex, the rate of nematode-hypha attachment did not appear to be influenced.

In A. oligospora (ATCC 24927) inhibitory effects of light on trap formation have been reported (Gronvold, 1989). Light did not affect nematode-hypha attachment or ring structure development in A. oligospora (CBS 289.82).

The trapping ability of hyphae in the isolate CBS 289.82 was retained even when the growth time of the hyphae was more than 70 days. Attachment of nematodes to hyphae from seven weeks old culture was comparable to that of four weeks old cultures. Mycelium, developing during six weeks at 25 °C and subsequently cultured for more than five weeks at 5, 10 or 15 °C, did not show any decline in attachment efficacy or development of trophic hyphae.

The following examples are intended to further illustrate the present invention, without limiting the scope of the invention.

3. Materials and Methods

The following disclosure also provides the technique how the nematodes can be trapped. The disclosure will then be completed with the description of the conditions under which the nematodes can be trapped, also merely by way of examples for non-limitative illustration purposes.

3.1 Legends to Figures

Fig.1 : Ability of five species of the Dactylaria-complex to capture Meloidogyne hapla and Meloidogyne incognita on CM A (1:10) at 25 °C. Entire bars represent % capture and the dark lower part the % immobilized nematodes. Error bars indicate standard deviation for capture.

Fig. 2: Ability of Arthrobotrys oligospora (CBS 289.82) fungi to capture

Meloidogyne hapla, Pratylenchus penetrans, Globodera pallida and Globodera rostochiensis. Open bar: % of nematodes captured by adhesive hyphae and surrounded by ring structures and filled with trophic hyphae. Error bars indicate standard deviation for capture.

Fig. 3: Capture mechanism of A. oligospora (CBS 289.82). Figure 3A: upper photo and 3B lower photo. Cryo microphotographs of second-stage juveniles of Meloidogyne hapla captured by means of adhesive hyphae (AH) and sometimes by adhesive coilings (AC) of A.oligospora. Multicellular ring structures (RS) develop after attachment of the nematodes to the hyphae. They did not necessarily develop at the places of initial attachment. A scale bar = 100 μm; B scale bar = 10 μm.

Fig. 4: Capture of second stage juveniles of Meloidogyne hapla by

Arthrobotrys oligospora (CBS 289.82). Juveniles of M. hapla (N=30) were followed individually while moving on the mycelial mat. Number of nematode-hyphae encounters was enumerated between start of the

experiment and attachment. Open bar: % of nematodes captured by adhesive hyphae; filled bar: % of non captured nematodes.

Fig. 5a: Ability of Arthrobotrys oligospora (CBS 289.82) to capture and infect second-stage juveniles of Meloidogyne hapla at different temperatures. In Exp. 1 carried out with four weeks old cultures at 25 °C followed by 2 days at the least temperatures. Exp. 2 as Exp. 1 but with six weeks old cultures. Open bar: % of nematodes captured by adhesive hyphae; dotted bar: % nematodes captured by adhesive hyphae and surrounded by ring structures; filled bar: % of nematodes captured by adhesive hyphae, surrounded by ring structures and at least filled with trophic hyphae. ¹ Different letters indicate significant differences between means of number of juveniles surrounded by ring structures within each experiment and each day (α = 0.05).

Fig. 5b: Ability of Arthrobotrys oligospora (CBS 289.82) to capture and infect second-stage juveniles of Meloidogyne hapla at different temperatures: Exp. 3 carried out with a culture grown for six weeks at 25 °C and followed by five weeks at the test temperatures. Exp. 4 carried out with a cultures grown for six weeks at the experimental temperatures. Open bar: % of nematodes captured by adhesive hyphae; dotted bar: % of nematodes captured by adhesive hyphae and surrounded by ring structures; filled bar: % of nematodes captured by adhesive hyphae, surrounded by ring structures and at least filled with trophic hyphae. ¹ Different letters indicate significant differences between means of numbers of juveniles surrounded by ring structures within each experiment and each day (α = 0.05).

Fig. 6: Development of trophic hyphae of Arthrobotrys oligospora (CBS

289.82) inside Meloidogyne hapla at different temperatures. Trophic hyphae development was quantified through estimation of the percentage of the nematode body length filled with trophic hyphae. Observed values are in markers, calculated values (Fieller-procedure in Genstat 5) in lines.

Fig. 7: Mobility of second-stage juveniles of Meloidogyne hapla on CMA

(1 :10, 1.5 % agar) at different temperatures. Different letters indicate significant differences between means (Analysis of variance followed by a Student's test, α < 0.05.

Fig. 8. Ability of Arthrobotrys oligospora (CBS 289.82) to capture and infect second-stage juveniles of Meloidogyne hapla on substrates with different nutrient levels: WA: water agar 1.5 %, CMA: corn meal agar 1 :10, LNM+ and LNM-: low nutrient mineral salts media respectively with and without 200 μgr/liter thiamin and 5 μgr biotin/liter. Open bar: % of nematodes captured by adhesive hyphae; dotted bar: % of nematodes captured by adhesive hyphae and surrounded by ring structures; filled bar: % of nematodes captured by adhesive hyphae, surrounded by ring structures and filled at least partly with trophic hyphae. Different letters indicate significant differences between means of % of juveniles surrounded by ring structures within each day (α = 0.05).

3.2 Fungal species and culture methods.

Stock cultures of Arthrobotrys conoides (CBS 265.83), Arthrobotrys dactyloides (CBS 109,37), Arthrobotrys oligospora (CBS 289.82), Arthrobotrys oligospora (CBS 115.81 =ATCC 24927), Arthrobotrys oligospora (ATCC 24927), Arthrobotrys scaphoides (CBS 226.52), Duddingtonia flagrans (CBS 565.50) and Monacrosporium cionopagum (CBS 228.52) were maintained on corn meal agar (Oxoid, CMA 1 :1, 1,5% agar) in Petri-dishes (diameter 8.8 cm) at 25 ± 1 °C, with monthly routine transfers to fresh medium.

3.3 Nematodes

A population of root-knot nematodes, M. incognita originally isolated from tomato plants in Dutch glasshouses, was obtained from the Department of Nematology, Wageningen Agricultural Unversity. M. hapla originally isolated from rose plants, was obtained from the DLO-Centre for Plant Breeding and Reproducing Research, Wageningen. The nematodes were maintained continuously on tomato plants (Lycopersicon esculentum Mill.cv Moneymaker) in silversand at a temperature of 22-25 °C and 20 °C respectively, in a greenhouse. Newly hatched second-stage juveniles were obtained by incubating egg masses on a 50 μm sieve in water for 2 days at 20 °C. The outer surface of 2-days old juveniles of Meloidgyne spp. was sterilized in a mixture of 0.02% (w/v) ethoxy-ethylmercury chloride (Aretan) and 0.1 % (w/v) streptomycin sulphate for about 2 h in a 10 ml conical centrifuge tube and subsequently washed three times in sterile water (s'Jacob & van Bezooyen, 1984). Because of possible changes in the nematodes surface or activity as a result of the sterilization procedure, which might influence the trapping ability of the fungi tested, in one test non-sterilized and water-washed juveniles of two different populations of M. hapla were included (isolated from rose and Bergenia sp.).

To assure purity of cultures, the Meloidogyne species were routinely characterized on the basis of enzyme phenotypes of females (Esbenshade & Triantaphyllou, 1985). Difference between species was based on their esterase, malate dehydrogenase and superoxide dismutase phenotypes.

A population (juveniles and adults) of the root lesion nematode, Pratylenchus penetrans (Cobb), was obtained from the Research Station for Floriculture, Aalsmeer; second-stage juveniles of the potato-cyst nematodes. Globodera rostochiensis (Wollenweber), and G. pallida (Stone) pathotypes Ro1 and Pa3, were obtained from a culture stock in the centre for Plant Breeding and Reproducing Research, Wageningen. These three nematode species were surface sterilized in streptomycin-Aretan mixture for 30 min in a Bϋchner funnel and subsequently

washed once in 0.1 % streptomycin and three times in sterile water. Contamination by microorganisms was regularly checked for by inoculating nematodes on bouillon agar or water agar.

3.4 Trapping ability

Individual 4-mm plugs cut from the periphery of a growing stock colony of A. conoides, A. dactyloides, A. oligospora (CBS 289.82, CBS 115.81 , ATCC 24927) A. scaphoides, D. flagrans and M. Cionopagum were placed upside down in the centre of small Petri-dishes (44 mm) on CMA 1 :10 (thickness 1-2 mm) and removed one week after inoculation. The Petri-dishes (Lux) had a coverglass bottom, thus facilitating observation with an inverted microscopic (Zeiss Axiovert 10 equipped with an enhanced contrast video system, Analogue Contrast Enhancement Zeiss). They were kept at 25 ± 1 °C for 21 more days. Subsequently, experiments were performed at 25 ± 1 °C, 90-100 % r.h., at pH = 6.5, without light (Waalwijk et al., 1990).

In each test, a drop adjusted to contain about 50 second-stage juveniles of M. hapla or M. incognita, was added to the 4-weeks old fungal cultures. Juveniles moved actively from the inoculation point. At 25 °C the mobility of second stage larvae was about 7.3 mm h ^"1 on CMA 1 :10, 1.5 % agar. At regular time intervals, the number of captured nematodes was counted and expressed as a percentage of the number of introduced nematodes. Mobile and immobile captured individuals were counted separately.

Assays usually consisted of 3 replicate Petri-dishes and three repetitions in time. Differences in percentage capture and immobilization were analyzed for days 1 , 2, 6 and 9 by performing one-way analysis of variance (ANOVA) on the arcsin- transformed values. Differences were further analyzed by comparing the means for the different fungi using Student's t-test.

Because D. flagrans and two isolates of A. oligospora did not show any ability to capture root-knot nematodes, experiments were repeated with younger fungus colonies (1, 2 and 3 weeks old). Tissue culture plates (24 wells of 15 mm diameter) were used because younger fungal colonies do not cover the whole agar surface of the small Petri-dishes.

Capture of four different plant-parasitic nematodes by Arthrobotrys oligospora (CBS 289.82). The ability of A. oligospora (CBS 289.82) to capture four different plant- parasitic nematode species: P. penetrans, G. rostochiensis (Ro1) and G. pallida (Pa3) and M. hapla was also tested using 4-weeks old fungal cultures in small Petri- dishes.

Total numbers of captured nematodes and the number of nematodes associated with the ring-structures and trophic hyphae were counted. Observations were made after 6 h, 1, 3, 6, 10 and 16 days. The experiment was repeated twice. Difference were analyzed by comparing the means using a generalized linear model (GLM) for binary data (Mc Cullagh & Nelder, 1989) followed by Student's t-test (RPAIR procedure of Genstat 5).

3.5 Capture mechanism

After it was demonstrated that A. oligospora (CBS 289.82) could capture nematodes with morphologically unmodified hyphae, 30 active second-stage juveniles of M. hapla were followed individually on the mycelial mat of a 4-week old culture from the moment of addition till capture by hyphae (maximum observation time 45 min). The number of nematode-hypha encounters was counted.

To check if nematodes were also captured by newly formed hyphae, 4-mm plugs cut from a stock colony were placed upside down in 24 well-plates on CMA 1:10 and removed 24, 48, 72, 96 f after inoculation.

The area which had been in contact with the mycelium from the plug was removed with a cork borer and subsequently filed up with fresh agar to guarantee that nematodes only could be captured by newly formed young hyphae. Six hours after addition of about 50 nematodes-fungus capture was checked.

3.5 Scanning electron microscopy

In order to ascertain whether ring structures developed at the initial point of attachment, the infection processes were studied on 20 second-stage juveniles of M. hapla.

Pieces of agar (4 mm in diameter and 1-2 mm deep, containing recently captured nematodes were mounted on copper stubs with a thin film of Tissue Tek (Hexland Ltd., England).

The samples were immediately frozen by immersion (plunge cooling) in nitrogen slush in the EMSCOPE SP2000 Cryogenic-Preparation System. The stubs were transferred under vacuum conditions to the preparation chamber where surface ice was etched by conductive heating. The specimens were subsequently sputtered with gold and transferred to the scanning electron microscope (Jeol JSM 35C), equipped with a cryo-stage. The specimens were photographed at 15 kV accelerating using a Kodak 35-mm Pan X film.

Because in A. oligospora (CBS 289.82) three dimensional structures develop after the nematode has adhered to the hypha, the term ring structures have been used in stead of capture structures.

3.6 Saprophytic growth rate

Experiments on saprophytic growth of several nematode-capturing fungi at different temperatures were performed in small Petri-dishes (diameter 44 mm) with those species which had trapped juveniles of Meloidogyne spp. in the earlier tests. Plugs of 2-week old cultures of A. conoides, A. dactyloides, A. oligospora (CBS 289.82), A. scaphoides and M. Cionopagum were inoculated on CMA 1 :10. Five Petri-dishes with each species were kept at temperatures of 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C.

The average mycelial growth was recorded as the average distance from the centre of the inoculated agar piece to the outer rim of the growing hyphae (at day 3 and day 7) and was expressed in mm per day.

Saprophytic growth was also measured for three isolates of A. oligospora: CBS 289.82, CBS 115.81 , ATCC 24927 (twelve replicates) at 25 °C. Hyphal density (mm ² mycelium per 100 mm ² area) was measured in two areas of each of 10 Petri- dishes for each isolate of A. oligospora using image analysis (GOP-302, Context Vision). Density was recorded by selecting an optimum threshold value for contrast line (hypha)-background (agar). The cultures were four weeks old and grown at 25

4. Examples

4.1 Ability of different nematophagous fungi to capture root-knot nematodes.

Both Meloidogyne spp. were captured with approximately the same efficacy by A. conoides, A. dactyloides, A. oligospora (CBS 289.82), A. scaphoides and M. cionopagum (Fig. 1). Other isolates of A. oligospora (CBS 115.81 and ATCC 24927) and D. flagrans (CBS 565.50) completely failed to capture juveniles from populations of M. hapla and M. incognita irrespective of the age of the mycelia (1 , 2, 3 or 4 weeks) or the sterilization method (streptomycin-Aretan, demi-water or

none). The surface treatment method did not affect the capture ability in the effective isolate of A. oligospora, CBS 289.82 and 100 % of the nematodes were captured in all treatments.

After, one day, significant differences could be observed between the five effective fungi both in percentage of nematodes captured (P< 0.001) and percentage of immobilized nematodes (P< 0.001)). A. oligospora (CBS 289.82) and M. cionopagum captured M. hapla and M. incognita to a significantly (P< 0.001) higher level (86 % to 96 % respectively) than A. conoides (51 % and 61 % respectively). Significantly fewer of these nematode species were captured in A. dactyloides and A. scaphoides: 0-25 % (P< 0.001).

Two days following the start of experiment A. scaphoides had caught significantly fewer juveniles of M. incognita than the other four fungi (P< 0.01). The proportion of nematodes captured by the other four fungal species reached a maximum between days 2 and 9 at values between 70 and 100 %. A conoides and A. scaphoides were less effective in immobilizating of nematodes than the other fungi (P< 0.001). Although by day 9, A. scaphoides had only captured 51 % of the M. incognita nematodes, most of those were immobilized, as in the more effective Meloidogyne-capturing fungi. In contrast, in the other less effective fungus A. conoides, 18 % of M. hapla nematodes and 37 % of M. incognita nematodes were still active after 9 days even when ring structures were present around the nematodes's body.

Capture of four different plant-parasitic nematodes by A. oligospora (CBS 289.82). Significant differences were observed in the ability of A. oligospora (CBS 289.82) to capture G. pallida, G. rostochiensis, M. hapla and P. penetrans, as well as in the formation of ring structures and the development of trophic hyphae in the interior of the various species (Fig. 2).

Of the four plant-parasitic nematode species tested, all the juveniles of M. hapla were captured within 6 h and those of P. penetrans (plus the adults) within 24 h. However, the number of juveniles of G. pallida and G. rostochiensis captured was significantly less and reached only about 30 % irrespective their activity. The formation of ring structure in A. oligospora (CBS 289.82) started with all nematode species within 24 h. After one day, 30 % of the juveniles of M. hapla had fungal ring structures around the body compared to only 10 % in the other nematodes species.

After sixteen days more than 90 % of the captured juveniles of M. hapla showed one or more ring structures around the body. The remaining 10 % of the nematodes laid motionless alongside the hyphae by which they had been captured. At the same time only 60 % of the nematodes of P. pentrans were surrounded by ring structures, 30 of those attached, continued to wriggle at the place of attachment, not yet inducing ring structures.

As for the two Globodera species tested, the development of ring structures lagged far behind: after 16 days only 27 % of the nematodes was surrounded by a ring, while the development of trophic hyphae inside the nematode body had not started at all. At day 16, more than 80 % of the nematodes of M. hapla were filled with trophic hyphae while in P. penetrans this percentage was below 40 % (Fig. 2). Development of trophic hyphae occured only when ring structures were present.

The data presented in Fig. 2 suggests that Globodera species were not normally colonised by the fungus in this test.

4.2 Capture mechanism of A. oligospora (CBS 289.82)

A. oligospora (CBS 289.82) showed the ability to capture nematodes by direct attachment to morphologically unmodified hyphae and to coilings, at places not markedly differentiated (Fig. 3A and B). Because this has not previously been

described for this species, its identity was confirmed twice by Centraal bureau voor Schimmelcultures, Baarn, The Netherlands, through identification of conidia developed on nematode-infested cultures. Observation of second-stage juveniles during locomotion showed that A. oligospora (CBS 289.82) attached to almost 45 % juveniles of M. hapla during one of the first contacts with a hyphae (Fig. 4). Average nematode-hypha encounters between the beginning of the experiment and attachment were about 100, two juveniles were not attached after 320 encounters with a hypha within the observation period of 45 min (Fig. 4). Nematodes were also attached to newly formed hyphae (24, 48, 72, 96 h) and these hyphae were able to produce ring structures around the nematodes.

Following attachment of the nematodes to the hyphae, 50 % of the ring structures did not develop at the initial attachment site (Fig. 3A and B). The subsequent development of an infection bulb and of trophic hyphae did not occur if ring structures were not found.

Saprophytic growth rate and hyphal density of several nematode-capturing fungi. As shown in Table 1 , A. conoides, A. oligospora (CBS 289.82) and M. cionopagum grew slowly at 5 °C but no growth was observed in the two other species. There was no growth of any species at 35 °C. Radial growth rate optima were at 15 - 20 °C or at 20 - 25 °C. A. scaphoides, the least effective capturing fungus, was the fastest growing species. Comparisons among A. oligospora isolates showed significant differences: at 15 °C, A. oligospora (CBS 289.82), the most effective capturing isolate, grew half as fast as the two other species and hyphal density was also significantly lower (Table 2).

Table 1 : Saprophytic growth rates of nematode-capturing Hyphomycetes on CMA 1:10 (mm/day)

A. conoides A. dacryl- A. oligo¬ A. sca¬ M. ciono¬

(CBS oides spora phoides pagum

265.83) (CBS (CBD (CBS (CBS 5109.37) 289.82) 226.52) 228.52)

Temp

5 0.1 ± 0.1 ¹ 0 0.1 ± 0.1 0 0.1 ± 0.3

10 0.4 ± 0.1 0.2 ± 0.1 0.4 ± 0.1 0.3 ± 0.1 0.2 ± 0.1

15 0.8 ± 0.1 2.3 ± 0.1 0.9 ± 0.4 2.0 ± 0.1 1.7 ± 0.6

20 1.7 ± 0.3 2.3 ± 0.2 2.3 ± 0.1 2.2 ± 0.3 2.1 ± 0.2

25 2.0 ± 0.3 1.7 ± 0.3 2.9 ± 0.1 5.0 ± 0.3 1.5 ± 0.2

30 2.0 ± 0.1 1.5 ± 0.2 2.5 ± 0.2 0.3 ± 0.2 0.3 ± 0.1

35 0 0 0 0 0

Mean ± s.e.

Table 2: Saprophytic growth rate (mm/day) and hyphae density (mm ² mycelium per 100 mm ² ) of three isolates of Arthrobotrys oligospora on CMA 1:1 at 25°C

Arthrobotrys oligospora

CBS 289.82 CBS 115.81 ATCC 24927

Saprophytic 2.9 ± 0.2 ¹ a ² 4.4 ± 0.3 b 4.3 ± 0.3 b growth

Hyphae density ³ 21.8 ± 0.4 ¹ a ² 23.5 ± 0.6 b 25.4 ± 0.6 c

1 Mean ± s.e.

² Different letters indicate significant differences between isolates (P < 0.001)

³ Hyphae density was measured using image analysis

4.3 Effect of temperature on nematode-hyphae attachment

In Table 3 an overview is given of variable parameters during incubation and testing in the various temperature experiments. Four or six weeks old cultures of A. oligospora grown at 25 °C (Experiments 1 and 2 respectively) were placed at each of the following constant temperatures, 48 h prior to the addition of nematodes: 5, 10, 15, 20, 25, 30 °C (and also 35 °C in Experiment 2) and tested at those temperatures.

Table 3: Experimental conditions of Temperature Experiments 1 to 5

time of culturing temperature during test fungus before culturing before temperature experiment experiment

(days) CQ CQ

Exp. 1 28 25 5, 10, 15, 20, 25, 30

Exp. 2 42 25 5, 10, 15, 20, 15, 30, 15

Exp. 3 42 + 25

36 5, 10, 15 5, 10, 15

Exp. 4 42 5, 10, 15 5, 10, 15

Exp. 5 28 15 5, 10, 15 n Experir πent 3, six weeks old fi jngal cultures of A. oligospo ra, grown at 25 °C and originating from the same batch as those in Experiment 2, were placed during more than five weeks at 5 °C, 10 °C or 15 °C and remained at these temperatures after addition of the nematodes (Table 3). In Experiment 4, cultures of A. oligospora

were grown during six weeks at 5, 10 or 15 °C and were tested at the same temperatures. In Experiment 5 the cultures were grown at 15 °C for four weeks and tested at 5, 10 and 15 °C (Table 3).

In Experiments 1 , 2 and 3 numbers of captured juveniles were counted daily starting after one day. Counts were expressed as a percentage of the total number of live nematodes counted immediately after the start of the experiment. Nematodes captured by hyphae, those surrounded by ring structures and the number of nematodes with trophic hyphae inside were scored separately. In Experiment 4 observations on capture started after 6 h, while in Experiment 5 the number of captured nematodes were registered already at 1 , 2, 3, 4 and 6 h.

At the end of each experiment the mycelial mat was examined for any morphological changes to check whether vegetative hyphae had died.

The proportion of nematodes captured by hyphae, surrounded by ring structures and filled with trophic hyphae were analyzed with a Generalized Linear Model (GLM) for binomial data (Mc Cullagh & Nelder, 1989), leading to an analysis of deviance for quantal data and subjected to a Student's t-test for pairwise comparison of treatments on a logit-scale. This was only possible if the mean proportions were not equal to 0 or 1. For these means significance was obtained by considering confidence intervals. Analysis were carried out with the Genstat computer programme (1987). All statistical tests were performed with a significance level of 0.05. In addition to variables mentioned above, in Experiment 2 also the colonization of each nematode was determined at regular intervals (day 1 , 2, 5, 8, 12, 15, 21, 27, 36, 44, 63) and expressed as percentage of body length filled with trophic hyphae.

Five classes were distinguished: 0 (no visible mycelium body), 0-25, 25-50, 50-75 and 75-100 % body length with hyphae. The products of the total percentage counts in each of the groups and the midpoints of these groups was summed to give a percentage estimate of the body length filled with trophic hyphae. The

technique is based on the assumption that the presence of trophic hyphae is indicative for the colonization. T ₅₀ and T _g5 (days after inoculation when 50 and 95 % of the nematode body length was filled with trophic hyphae) were calculated under the assumption of logistic growth increase of trophic hyphae filled body length. T ₅₀ and T ₉₅ were estimated for each Petri-dish and subsequently analysed by analysis of variance followed by student's t-test for pairwise comparison of treatments.

Figure 5a and 5b show the effects of temperature on the ability of hyphae of A. oligospora (CBS 289.82) to capture and infect M. hapla. At the first observation 24 h after inoculation of the nematodes, all second-stage juveniles of M. hapla were captured by the hyphae of the four weeks old cultures of A. oligospora at all temperatures below 35 °C (Experiment 1 and 2). Nematodes became attached at any part of the body. At 35 °C, nematodes and hyphae failed to attach to each other.

Mycelium of A. oligospora grown under suboptimal temperatures for vegetative growth (Experiment 3 and 4, Fig. 5b), did not show any decline in nematode attachment compared to hyphae grown at the optimum temperature for vegetative growth (Experiments 1 and 2, Fig. 5a). In experiment 4, all nematodes were captured at the first observation after six hours, irrespective of the temperatures tested (Fig. 5b). Immediate observation one hour after the start of Experiment 5 showed that all nematodes were captured at the temperatures tested.

4.4 Effect of temperature on ring structure development and effect of temperature on activity of second-stage juveniles of M. hapla

Nematode activity was measured in order to analyse whether any temperature effects on the capture of nematodes was due to the ability of the fungus to capture juveniles of M. hapla or to the activity of the juveniles. In the centre of a Petri-dish with CMA 1:10, 1.5 % agar, 50 axenic second-stage juveniles of M. hapla (from the same batch as in Experiment 2) were inoculated. Two hours after introduction, nematode mobility was assessed by counting the number of juveniles in four concentric zones of 5 mm from the inoculation point. Tests were performed at 5 °, 10 °, 15 °, 20 °, 25 °, 30 ° and 35 °C (see table 4).

Table 4: Development of trophic hyphae of Arthrobotrys oligospora (CBS 289.82) in second-stage juveniles of Meloidogyne hapla at different temperatures, expressed in days after inoculation when 50 % and 95 % of the nematode body length was filled with trophic hyphae (T ₅₀ and T _g5 ).

Temperature ^T 50 ^T 95 CQ

5 47.6 ± a ² 77.2 ± 8.9 a 3.8 ¹

10 24.8 ± 1.0 b 35.4 ± 4.7 b

15 15.1 ± 1.8 c 28.3 ± 2.8 be

20 10.6 ± 0.8 cd 18.5 ± 0.3 cd

25 7.9 ± 0.3 d 13.4 ± 1 ,1 d

30 7.8 ± 0.9 d 13.5 ± 0.7 d

35 α> n.t. ∞ n.t.

¹ Mean ± s.e.

² T ₅₀ and T _g5 were estimated for each Petri-dish and subsequently tested by analysis of variance followed by Student's t-test for pairwise comparison of treatments. Different letters indicate significant differences between means in the column (α < 0.05). n.t.: not tested

For each temperature an average mobility was calculated using the following formula: i=3 M = ∑ f. Di i = u f = fraction of nematodes present in area i Di = average distance (mm) from centre for area i = 0 0 < r < 5 mm Di = 5 mm

= 1 5 < r < 10 mm Di = 7.5 mm

= 2 10 < r < 15 mm Di = 12.5 mm

= 3 15 < r < rim Petri dish Di = 17.5 mm The nematode mobility for different temperatures was analysed by using one-way ANOVA, followed by a Student's t-test for comparison of means.

Subsequent development of ring structures around the nematodes differed significantly between tested temperatures during the first six days after capture (Experiment 1, Fig. 5a). At 5 and 10 °C only 2 % of the nematodes became surrounded, at 15 °C the number of nematodes surrounded by ring structures was 15 % and at 20, 25 and 30 °C this number was significantly higher, namely 50 %. At day 2 these differences were still significant although less pronounced. At 5 and 10 °C the number of surrounded nematodes reached only 20 %, at 15 °C this number reached about 60 % and at three highest temperatures tested, the proportion of nematodes surrounded by ring structures reached almost 100 %. At day 8 at all

temperatures the ring structure development was 90 % or higher.

In the higher temperature experiment the development of ring structures around nematodes progressed more rapidly (Fig. 5a). After one day, almost all nematodes were surrounded by ring structures at 15 °C, whereas at 5 and 10 °C these number were less than 40 %. At days 7 and 8 all nematodes were surrounded at all temperatures < 30 °C even when the mycelium was grown at low temperatures (Experiment 3 and 4, Fig. 5b).

4.5 Effect of temperature on trophic hyphae development

Development of trophic hyphae in the nematode body was significantly influenced by temperature. This resulted in significant differences in the number of days after which 50 % or 95 % of the nematode body length was filled with hyphae (Fig. 6, Table 5). In mycelium of A. oligospora, grown or kept under suboptimal conditions for vegetative growth (Experiment 3 and 4), development of trophic hyphae was not altered in comparison to the earlier experiments (Fig. 5a and b).

Table 5: Growth of A. oligospora (CBS289.82) on different substrates before and after additionof second-stage juveniles of Meloidogyne hapla.

Medium Vegetative hyphae Number of ring structures

PH Growth ¹ Density ² Before After Number of ring structures addition of M. addition of around M. hapla ⁴ hapla ³ M. hapla ³

1.5 % water 5.5 2.7 ± 0.1 a ⁵ 23.2 ± 1.5 ⁵ a 5 4 2.8 ± 1.3 ⁵ a agar

LNM- ⁶ 6.5 3.1 ± 0.1 b 19.9 ± 2.8 b 3 2 4.0 ± 1.8 b ro

LNM+ ⁶ 6.5 3.4 ± 0.1 c 20.7 ± 2.6 b 2 2 4.6 ± 1.6 b oo c

CMA 1 :10 5.5 2.9 ± 0.1 a 21.5 ± 1.5 c 1 1 2.5 ± 1.4 a b

Radial growth: mm/d ay

² Mycelial density: mm ² mycelium per 100 mm ² agar in four weeks old fungal colonies

³ Number of ring structures in 20 fields of sight (at 200x magnification) Number of ring structures around 10 nematodes per Petri dish one day after addition of the nematodes

⁵ Mean ± s.e. Different letters indicate significant differences (Student's t-test. α = 0.05)

⁶ Low nutrient mineral salts medium with(+) and without(-) thiamin and biotin

4.6 Nematode mobility in response to temperature

All tested juveniles of M. hapla dispersed over the agar. The nematode activity is shown in Fig. 7. Mobility differed significantly between the temperatures tested: at 25 °C the highest mobility was reached, 7.3 mm h ^"1 , while at 5 °C a mobility of 2.2 mm h ^"1 was observed (Fig. 7).

4.7 Effect of growth substrates on capture ability

The fungus was grown on substrates differing in nutritional quality: water agar 1.5 % corn meal agar (CMA, 1:10) and a low nutrient mineral salts medium with or without 200 μgr thiamin and 5 μgr biotin/liter (LNM + and LNM- respectively, Nordbring-Hertz, 1973). One and four weeks old fungal cultures of A. oligospora grown at 25 °C, were inoculated with 50 axenic second-stage juveniles of M. hapla at 25 °C. Observations on number of attached and infected nematodes were made 1 and 6 h and 1, 2, 6 and 16 days after addition of nematodes. The average number of ring structures around the attached nematodes counted one day after the start of the experiment. Each treatment consisted of 3 replicates and the experiment was repeated three times (Fig. 8).

At day 6 and 14 the development of trophic hyphae in the interior of the nematode body reached about 55 % and 85 % in LNM-, CMA and LNM + . On WA these percentages were significantly lower: in only 25 % and 50 % of the nematodes trophic hyphae developed. The number of captured nematodes, nematodes surrounded with ring structures and nematodes with trophic hyphae were analysed as described for the temperature experiments.

In order to analyse if differences in capture of nematodes were due to higher frequency of nematodes encountering hyphae and or ring structures, mycelium growth, hyphal density and numer of spontaneous ring structures (freely and not surrounding nematodes) were determined. Average mycelial growth rate (mm/day)

was recorded as the average distance from the inoculated agar piece to the outer rim of the growing hyphae (at day 3 and 5 in 10 replicates). The hyphal density, expressed as area of mycelium mm ² per 100 mm ² was measured in two areas of 10 Petri-dishes for each nutrient level in the four weeks old colonies, using image analysis (GOP-302, Context Vision). Ring structure formation was observed one day before and one day after addition of nematodes, by counting the number of ring structures in 20 fields of sight (at 200x magnification).

Within one hour following addition of nematodes, all second-stage juveniles of M. hapla were captured successfully by the hyphae of A. oligospora, irrespective of differences in hyphal density at the various nutrient levels (Table 5). One day after the start of the experiment a higher percentage of nematodes was surrounded by ring structures on LNM + (76 %) as compared to LNM- and CMA 1:10 (61.0 and 63 %, respectively), while WA demonstrated a significantly lower percentage of juveniles surrounded by ring structures (39 %). After two days this percentage increased to 84 % on WA and to about 97 % on the other substrates. The number of ring structures around the nematodes was significantly higher on LNM+ and LNM-, than either on water agar or corn meal agar (Table 5).

4.8 Effect of light on capture ability

Four weeks old cultures of A. oligospora were inoculated with 50 axenic second- stage juveniles of M. hapla and incubated ant 25 °C. Three Petri-dishes were placed in the dark and three Petri-dishes were incubated under constant artificial light (Philips Pis lamp 11 Watt, wavelength 310-765 nm, 8.6 J/cm ² /h). Numbers of captured and infected nematodes were counted at regular intervals (day 1, 2, 4, 7, 9, 11, 14, 23). The number of ring structures surrounding juveniles was counted 3 and 13 days after the start of the experiment. Per Petri-dish ten randomly selected fields of sight were examined (at 200x magnification). Only completely closed ring structures were enumerated. In one experiment two isolates of A. oligospora (ATCC

24927 and CBS 115.81) were included under the same experimental conditions. As the results of all three experiments on the influence of light (8.6 Joule/cm ² /h, 310- 765 nm) were alike, results are given for only one (Table 6). Light did not alter the number of nematodes captured by A. oligospora nor had light a significant effect on the number of ring structures induced or the development of trophic hyphae in nematodes.

Table 6: Capture ability of Arthrobotrys oligospora (CBS 289.82) after addition of second-stage juveniles of Meloidogyne hapla in continuous (310-765 mm, 8.6 J/cm ² /h) or darkness. (Tests performed in triplicate)

N n Tr

Days % juveniles attached to % juveniles surrounded by % juveniles filled with adhesive hyphae ring structures trophic hyphae

Light Darness Light Darkness Light Darkness

1 100 ± o ¹ 100 ± 0 a 62 ± 7.7 62.2 ± a 0 ± 0 0 ± 0 a 4.4

2 100 ± 0 100 ± 0 a 88.5 ± 88.3 ± a 0 ± 0 0 ± 0 a 4.8 4.5

4 99.4 ± 100 ± 0 a 91.1 ± 94.4 ± a 6.1 ± 2.8 6.6 ± 1.8 a 0.9 3.1 0.5 ω l\)

7 98.8 ± 100 ± 0 a 95.0 ± 96.2 ± a 39.5 ± 19.9 49.8 ± a 1.7 4.4 1.8 3.9

9 98.7 ± 100 ± 0 a 90.5 ± 98.6 ± a 56.4 ± 12.4 71.0 ± a 1.7 5.1 2.0 3.8

14 98.1 ± 100 ± 0 a 90.6 ± 98.6 ± a 71.0 ± 10 85.0 ± a 1.5 4.0 2.0 1.6

23 98.0 ± 100 ± 0 a 91.2 ± 98.6 ± a 75.9 ± 8.2 88.8 ± a 1.5 3.1 2.0 2.5

1 Mean ± s.e. 2 Different letters indicate significant differences between means in the column (α = 0.05). The number of ring structures surrounding juveniles counted 3 and 13 days after the start of the experiments, were not influenced significantly by light or dark (at day 3:3.3 and 3.6 and at day 13, 3.6 and 3.7 respectively). Also, light did not alter A. oligospora (ATCC 24927) and A. oligospora (CBS 115.81): second-stage juveniles of M. hapla did not attach to the fungus.

4.9 Effect of ageing of fungal mycelium on capture and infection.

Experiments on the effects of the growth time of the fungal mycelium on the capture and infection of nematodes were conducted as parts of the experiments on the temperature and nutrient level by using fungal varying in age.

Seven week old cultures of A. oligospora did not show a decline in capture and infection of nematodes in comparison to four week old cultures (Experiment 2 and 1 respectively, Fig. 5a).

Capture ability of A. oligospora and subsequent infection by trophic hyphae did not show any decline after keeping mycelium at low temperatures during a prolonged period of time (Experiment 3, 78 days: 42 days at 25 °C and subsequently 36 days at 5, 10 or 15 °C Experiment 4, 42 days at 5, 10, 15 °C). In this respect there was no significant difference with Experiment 1. In none of the experiments dead or morphologically aberrant hyphae were found in the time course of the experiments.

Under adverse temperatures that do not favour ring structure development or vegetative growth, or under poor nutritional conditions for ring structures development, our isolate tested shows the capacity to attach nematodes with adhesive hyphae. This clearly illustrates lower demands on temperature and nutrition than needed for ring structure formation. This implies that the range of circumstances for this fungus to capture nematodes and to be active as a control agent is very broad.

5. References

Belder, E. den (1991). Screening for the activity of fungal metabolites. In: Methods for studying nematophagous fungi. Kerry, B.R. & Crump, D.H. (Eds.). lOBC/WPRS Bulletin, pp. 47-57.

Blackburn, F. & Hayes A. (1966). Studies on the nutritions of Arthrobotrys oligospora Fresn. and A. robusta Dudd., I. The saprophytic phase. Ann. Appl. Biol. 58: 43-50.

Boag, B., Robertson, W.M. & Ainsworth, L.F. (1988). Observations on the specificity of the nematophagous fungus Arthrobotrys dasguptae (Shome and Shome) to plant parasitic nematodes. Nematologica 34, 238-245.

Cayrol, J-C. et al. (1993). La Recherche, vol 24, no 250, January 1993, FR pages 78-80. Les biopesticides a I'assaut des nematodes du sol'.

Couch, J. N. (1937). The formation and operation of the traps in the nematode- catching fungus, Dactylella bembicoides. J. Elisha Mitchell Sci. Soc. 53: 301-309.

Esser, R. P., El-Gholl, N. E. & Price, M. (1991), Biology of Dactylella megalospora Drechs., a nematophagous fungus. Soil Crop Sci. Sco. Fla. Proc. 50, 173-180.

Feder, W. A. (1963). A comparison of nematode-capturing efficencies of five Dactylelia-species at four temperatures. Mycopath. Mycol. Appl. 19: 99-104. Genstat 5 Committee (1987). Genstat 5 reference manual, Clarendon Press, Oxford.

Gronvold, J. (1989). Induction of nematode-trapping organs in the predacious fungus, Athrobotrys oligospora (Hyphomycetales) by infective larvae of Ostertagia ostertagi (Trichostrongylidae). Acta Vet. Scand. 30: 77-87.

Hayes, W. A. & Blackburn F. (1966). Studies on the nutrition of Arthrobotrys oligospora Fres. and A. robusta dudd., II. The predacious phase. Ann. App. Biol. 58: 51-60.

Heintz, C. E. (1978). Assessing the predacity of nematode-trapping fungi in vitro.

Mycologia, 70: 1086-1100.

s'Jacob, J.J. & van Bezooyen, J. (1984). In: A manual for practical work in nematology (revised edition). Int. Postgraduate Nematology Course, Int. Agric. Centre, Wageningen, The Netherlands.

Jaffee, B.A., Muldoon A. E. & Tradford, E. C. (1992). Trap production by nematophagous fungi growing from parasitized nematodes. Phytopathology 82: 615-620.

Jansson, H. B. & Nordbring-Hertz, B. (1980). Interactions between nematophagous fungi and plant-parasitic nematodes: attraction, induction of trap formation and capture. Nematologica 26: 383-389.

Leach, C. (1971). A practical guide to the effects of visible and ultraviolet light on fungi. In: Methods of microbiology, Booth, C. (Ed). Academic Press, London, pp. 609-664.

McCullagh P. & Nelder, J. A. (1989). Generalized linear models (2nd ed). Chapman and Hall, London, England.

Murray D. S. & Wharton D. A. (1990). Capture and penetration of the free-living juveniles of Trichostrongylus colubriformis (Nematoda) by the nematophagous fungus Arthrobotrys oligospora. Parasitology 101: 93-100.

Nordbring-Hertz, B. (1968). The influence of medium composition and additions of animal origin on the formation of capture organs in Arthrobotrys oligospora. Physiol. Plant. Pathol. 21: 52-65.

Nordbring-Hertz, B. (1973). Peptide-induced morphogenesis in the nematode- trapping fungus, Arthrobotrys oligospora. Physiol. Plant 29: 223-233.

Nordbring-Hertz, B. (1977). Nematode-induced morphogenesis in the predacious fungus, Arthrobotrys oligospora, Nematologica 23: 443-451.

Nordbring-Hertz, B. Stalhammar-Carlemalm. M. (1978). Capture of nematodes by Arthrobotrys oligospora, an electron microscopic study. Can. J. Bot. 56: 1297-1307.

Poinar, G. O. & Jansson, H. B. (1986). Infection of Neoplectana and Heterorhabditis (Rhabditida: Nematoda) with the predatory fungi, Monacrosporium ellipsosporum and Arthrobotrys oligospora (Moniliales: Deuteromycetes). Revue Nematol. 9: 241-244.

Stirling, G. R. (1991). Biological control of plant parasitic nematodes: progress, problems and prospects. C. A. B. International, Wellington, pp. 282.

Soprunov, F. F. (1966). Predacious hyphomycetes and their application in the control of pathogenic nematodes. (Translation from Russian) Israel program for scientific translations, Washington, p. 292.

Tunlid, A., Johansson, T. & Nordbring-Hertz, B. (1991). Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora, J. Gen. Microb. 137, 1231- 1240.

Tunlid, A., Jansson, H.B. & Nordbring-Hertz, B. (1992). Fungal attachment to nematodes. Mycol. Res. 96, 401-412.

Veenhuis, M. Sjollema, K., Nordbring-Hertz, B. & Harder, W. (1989). An improved method for light-and electron microscopical studies of nematode/fungal interactions. Antonie van Leewenhoek 55: 361-368.