S. A. Henriksen

In vitro stress selection of nematophagous fungi for biocontrol of parasitic nematodes in ruminants.

Laboratory experiments were designed to select nematophagous fungi that were able to survive in vitro conditions simulating passage through the gastro-intestinal tract of cattle. All of the tests were conducted at 39 degrees C. In a primary stress selection step in diluted rumen fluid, 21 isolates were obtained. Each of the primary stress selected isolates was tested in synthetic saliva, rumen fluid simulating the activity in the rumen, rumen fluid followed by pepsin-hydrochloric acid treatment simulating the additional effect of ruminal and abomasal activity, pepsin-hydrochloric acid solution simulating conditions in the abomasum and finally in a trypsin solution as an example of enzyme activity in the gut. The effect of the rumen fluid alone, or rumen fluid followed by pepsin-hydrochloric acid treatment, were responsible for the reduction in surviving fungal isolates. Only six of thirteen isolates belonging to the genus Arthrobotrys survived while seven of eight isolates of the genus Duddingtonia survived. Fourteen isolates were tested for their predatory capacity in a dung pat bioassay. Fungi of the genera Arthrobotrys and Duddingtonia reduced the development of Ostertagia ostertagi third stage larvae by approximately 75% and 96% respectively compared to the number of larvae that developed from fungus-free control pats.

In vivo passage through calves of nematophagous fungi selected for biocontrol of parasitic nematodes

The experiment was designed to test the survival and performance of stress selected nematophagous fungi after passage through the gastro-intestinal tract of cattle. Ruminating calves were fed daily with a fixed amount of fungal material grown on barley grains. The excreted dung was collected on days four and five after the start of the feeding experiment. Barley grains were washed out of the excreted dung and incoculated on water-agar plates. After incubation for one week, nine of ten fungal isolates were re-isolated from these plates. The predatory capacity of the fungi in the excreted faeces was tested in a dung pat bioassay and a faecal culture system. In the dung pat bioassay, two fungi of the genus Arthrobotrys and six of the genus Duddingtonia reduced the development of Ostertagia ostertagi third stage larvae by 85% (61%-93%), compared to the number of larvae developed from fungus-free control pats. In seven out of these eight isolates, the reduction of larvae in the faecal cultures was 92% (76%-99%).

Induction of traps by Ostertagia ostertagi larvae, chlamydospore production and growth rate in the nematode-trapping fungus Duddingtonia flagrans

Biological control of parasitic nematodes of domestic animals can be achieved by feeding host animals chlamydospores of the nematode-trapping fungus Duddingtonia flagrans. In the host faeces, D. flagrans develop traps that may catch nematode larvae. In experiments on agar, D. flagrans had a growth rate between 15 and 60 mm/week at temperatures between 20 and 30 degrees C. The presence of nematodes induces the fungus to produce traps. The rate of trap formation in D. flagrans has an optimum at 30 degrees C, producing 700-800 traps/cm2/2 days, when induced by 20 nematodes/cm2 on agar. Approaching 10 and 35 degrees C the ability to produce traps is gradually reduced. The response of chlamydospore production on agar to changes in temperature is the same as that for trap formation. On agar, at 10, 20 and 30 degrees C D. flagrans loses its trap inducibility after 2-3 weeks. During the ageing process, increasing numbers of chlamydospores are produced up to a certain limit. The time for reaching maximum chlamydospore concentration coincided with the time for loss of induction potential. The implications of these results in relation to biological control in faeces are discussed.

Biological control of Ostertagia ostertagi by feeding selected nematode-trapping fungi to calves.

Three nematode-trapping fungi, one Arthrobotrys oligospora and two Duddingtonia flagrans isolates, were fed to Ostertagia ostertagi-infected calves to test their ability to destroy larvae of this parasite in faeces and consequently to reduce the transmission of infective larvae to herbage. The fungi had previously been selected for their capability to pass the alimentary tract of cattle without losing growth and nematode-trapping potentials. Dung was collected from three calves each fed one of the three fungi and placed as 1-kg cow pats on a parasite-free grass plot together with control cow pats from a calf that was not given fungi. The cow pats contained comparable concentrations of parasite eggs. The two D. flagrans isolates were highly effective in that they reduced herbage larval infectivity by 74-85%. In contrast, A. oligospora did not show any effect in the present experiment. Field experiments will demonstrate if D. flagrans represents a potential organism for biological control of bovine gastrointestinal nematodes under practical agricultural management conditions.

An attempt to implement the nematode-trapping fungus Duddingtonia flagrans in biological control of trichostrongyle infections of first year grazing calves.

An attempt was made to control Ostertagia ostertagi by feeding the nematode-trapping fungus Duddingtonia flagrans (DSM 6703) to grazing calves. One group of calves (group E) was fed the fungal material in the first two months of the grazing season while another group was a non-treated control group (group C). Group E showed significantly lower faecal egg count in August and September. On four occasions in July and September, the herbage larval counts were significantly lower on the plot with the fungal-treated group than those recorded on the control plot. The average abomasal larval and adult worm counts were significantly reduced in August in group E and the average total worm count in the abomasum of group E was reduced by 87% in August compared to the non-treated group C. In October, the difference in average abomasal worm counts between group E and C was insignificant. Due to weight loss at the end of the grazing season, the control group showed a significantly lower average weight increase.

Laboratory experiments to evaluate the ability of Arthrobotrys oligospora to destroy infective larvae of Cooperia species, and to investigate the effect of physical factors on the growth of the fungus

Laboratory investigations were designed to study the influence of temperature, pH and oxygen tension on the growth of Arthrobotrys oligospora, a nematode-trapping microfungus. Experiments were performed to evaluate the potential role of A. oligospora in destroying third-stage larvae of Cooperia spp. on agar plates and in cattle faeces. The fungus had a growth rate optimum at 23 degrees C and pH 6. Anaerobic cultivation for 23 hours at 23 degrees C and 39 degrees C inhibited fungal growth, but it did not destroy the fungus, which regained growth upon a subsequent shift to aerobic conditions at 23 degrees C. Under experimental conditions in petri-dishes containing agar, the nematode-trapping efficiency of the fungus was striking in that 100% of a population of third-stage larvae of Cooperia spp. was captured within three days of the experiment. The trapping efficiency in faeces was shown to depend upon the inoculation level. At a concentration of approximately 2500 conidia per g faeces, 99% of the larvae were destroyed. The possibilities of using nematode-trapping fungi in controlling animal-parasitic nematodes are discussed.

Attempts to control infection with Ostertagia ostertagi (Trichostrongylidae) in grazing calves by adding mycelium of the nematode-trapping fungus Arthrobotrys oligospora (Hyphomycetales) to cow pats.

Artificially prepared cow pats containing Ostertagia ostertagi eggs were deposited on two comparable grazing plots at weekly intervals during June and July 1987. Before deposition the cow pats, on the one plot, were inoculated with 0.250 g mycelial fragments per kg of faeces of the predacious fungus Arthrobotrys oligospora. On the other control plot comparable numbers of non-inoculated cow pats were placed at the same time. Subsequently, in July, two equal groups of calves were turned out one on each plot to graze for two months after which they were necropsied. The number of infective O. ostertagi larvae in inoculated cow pats was reduced by 42% and herbage larval infectivity around them by 50-71% as compared with the corresponding parameters in control pats and surrounding herbage. After a grazing period of two months the calves on the plot with inoculated cow pats had acquired worm burdens that were on average 37% lower than those of calves on the control plot. The lower degree of parasitism in these calves was also reflected in lower egg counts, lower serum pepsinogen levels and higher body weight.

The capability of the predacious fungus Arthrobotrys oligospora (Hyphomycetales) to reduce numbers of infective larvae of Ostertagia ostertagi (Trichostrongylidae) in cow pats and herbage during the grazing season in Denmark

Artificially prepared cow pats containing Ostertagia ostertagi eggs were deposited on two pasture plots in May, June and July 1986. Half of the cow pats, placed on one plot, were inoculated with 2000 conidia per gram faeces of the predacious fungus Arthrobotrys oligospora. On the other plot fungus-free control cow pats were placed at the same time. In the faeces generally fewer infective O. ostertagi larvae developed in the inoculated than in the control cow pats. On the herbage around the control cow pats deposited in May, June and July a maximum concentration of infective larvae was found at the same time on the 6th of August 1986. At that time the herbage larval infectivity around inoculated cow pats deposited in May, June and July was subject to a reduction of 48%, 89% and 46%, respectively, compared with fungus-free control cow pats. This experiment indicates that a concentration of 2000 A. oligospora conidia per gram faeces results in a significant lowering of the herbage larval infectivity during the grazing season in Denmark.

Field experiments on the ability of Arthrobotrys oligospora (Hyphomycetales) to reduce the number of larvae of Cooperia oncophora (Trichostrongylidae) in cow pats and surrounding grass

In field experiments, conducted on parasite-free grass plots in two consecutive summers, artificially prepared cow pats containing Cooperia oncophora eggs were inoculated with the nematode-trapping fungus Arthrobotrys oligospora. Numbers of infective C. oncophora larvae isolated from the pats as well as from the surrounding herbage were subject to an approximately ten-fold reduction as compared with numbers in fungus-free pats and herbage surrounding these. This reduction was undoubtedly a result of entrapment of the parasite larvae within the faecal pats.

Biotic and abiotic factors influencing growth rate and production of traps by the nematode-trapping fungus Duddingtonia flagrans when induced by Cooperia oncophora larvae

A series of experiments on corn meal agar was carried out to evaluate the efficacy of the nematode-trapping fungus Duddingtonia flagrans in different abiotic and biotic conditions which occur in cow pats. Above a concentration of 50 parasitic larvae (L 3 ) cm –2 the fungus produced a maximum of between 500 and 600 nets cm –2 at 20°C in 2 days on the surface of corn meal agar. There were no differences in the trap-producing capacity of three strains of D. flagrans (CIII4, CI3 and Trol A). On agar at 30° and 20°C, the fungus responded to Cooperia oncophora L 3 very quickly producing a maximum of trapping nets 1 day after induction. At 10°C, traps were produced slowly starting on day 4 after induction and continued over the following week. Duddingtonia flagrans (CI3) grew at a normal rate at least down to an oxygen concentration of 6 vol.% O 2 , but it did not grow anaerobically. On agar, D. flagrans (CI3) did not produce trapping nets in an anaerobic atmosphere. Moreover, C. oncophora L 3 stopped migration under anaerobic conditions. When the fungal cultures were transferred to a normal aerobic atmosphere, after 1 and 2 weeks under anaerobic conditions, the C. oncophora L 3 resumed migrating on the agar and, in response, D. flagrans produced traps in the same amount as when it had not been under anaerobic stress. Under microaerophilic conditions (6 vol.% O 2 ) D. flagrans was able to grow, but the C. oncophora L 3 were not able to induce trapping nets in D. flagrans (Trol A) because of larval immobility. But, as under anaerobic conditions, the fungus could return to a nematode-trapping state when transferred to a normal aerobic atmosphere within 1 or 2 weeks if migrating nematodes were present. Under natural conditions in the cow pat it is expected that the fungus will be ready to attack parasitic larvae, when the oxygen tension increases as a result of, for example the activity of the coprophilic fauna. Artificial light giving 3000–3400 Lux on the surface of the agar significantly depressed the growth rate and the production of trapping nets in D. flagrans (CI3). On agar, D. flagrans (CI3) could grow and produce trapping nets at pH levels of 6.3 to 9.3. Net-production has its optimum between pH 7 and 8. On dry faeces mycelial growth was 7–10 mm during a 15 day period while on moist faeces the fungus expanded 15–20 mm during the same period. Based on the parameters investigated, D. flagrans is expected to be especially active in the well aerated surface layer of a cow pat, an area which normally contains a high concentration of infective nematode parasite larvae, but also an area where the temperature can be high and the water content low.