Granular formulations of the nematophagous fungus Arthrobotrys dactyloides were developed by incorporating fungal biomass into sodium alginate. The fungus was mass-produced by submerged fermentation in a broth containing glucose, peptone and yeast extract (GPY). Mycelia were blended with sodium alginate and the solution was allowed to drip into aqueous calcium gluconate to form granules. Some batches of granules were dried immediately, some were re-fermented in GPY for an additional 2 days, and various organic nutrients were incorporated into other batches before they were granulated. The granules produced in this manner were free of contaminants, had good fungal viability and vigour and remained viable when stored in vacuum sealed plastic bags at 4°C for 2 years. When granules were introduced into field soil, the fungus proliferated from granules, produced constricting ring traps and showed activity when tested against Meloidogyne javanica. The addition of organic matter to granules did not have any beneficial effect on A. dactyloides, whereas re-fermentation of granules improved the performance of the fungus. Re-fermented granules without organic matter were therefore used to study the behaviour of the fungus following its introduction into soil.
Trap formation by A. dactyloides was strongly stimulated by the presence of nematodes, or by substances associated with nematodes. Stimulatory effects were greater with more mobile nematodes (e g. Caenorhabditis elegans) than with M. javanica. A. dactyloides readily trapped nematodes that were present in soil, with trapping increasing as the number of nematodes increased, suggesting that nematophagous activity was density-dependent. Soil microorganisms also stimulated ring formation and trapping activity. Few rings were formed in pasteurised soil in which nematodes but not microorganisms had been removed by heating to 70°C for 1 hour, whereas no rings were produced in sterile soil. A. dactyloides did not produce traps on agar plates, but trapping activity increased significantly in the presence of microorganisms.
When inoculated onto media with different C/N ratios, A. dactyloides did not grow on materials with a C/N ratio less than 15. Addition of organic matter to soil did not have a direct effect on ring formation or trapping activity. However, it had an indirect effect by influencing populations of microorganisms and free-living nematodes, thereby stimulating the activity of A. dactyloides.
Environmental factors such as soil temperature, soil moisture and soil texture influenced ring formation and trapping activity. A. dactyloides grew from granules at temperatures as low as 12°C and its optimum temperature for growth was about 27°C. Temperatures greater than 35°C were lethal. Mortality of both M. javanica and C. elegans due to trapping by A. dactyloides was greatest at temperatures of 27 - 30°C, which suggested that biological control activity was greatest at temperatures which are ideal for both the fungus and its nematode prey. A. dactyloides grew best in soil with a
moisture content near field capacity, but survived extremely dry and wet conditions for at least 1 week. More rings were formed as the moisture content of dry soil was increased to field capacity, and this was reflected as increased nematode mortality. Furthermore, A. dactyloides produced more rings and trapped more nematodes in a fine-textured soil than in a coarsely-textured soil, possibly because it supported higher populations of nematodes and microorganisms and was less susceptible to fluctuations in soil moisture content.
When introduced into field soil as a granular formulation, A. dactyloides readily grew from granules, produced rings on all parts of the mycelia and trapped nematodes for at least 40 days. The length of trapping activity depended upon the availability of nematodes as a food source, as it was extended significantly by adding free-living nematodes or by increasing their population density by amending soil with organic matter. The fungus used trapped nematodes as a source of nutrients and grew from infected nematodes to trap more nematodes.
A. dactyhides grows about 0.9 mm/day, whereas tomato roots grow about 4 mm/day. When granules containing the fungus were placed adjacent tomato seed, the fungus was not consistently detected on parts of the root only 1 cm from the seed. Also, the fungus was rarely detected in the rhizosphere when tomato seedlings were grown in soil heavily infested with the fungus. Thus A. dacty hides was not rhizosphere-competent. However, A. dactyloides proliferated well in soil and 10 days after being introduced, mycelia bearing ring traps had extended about 8 mm from each
granule. This suggested that it may be possible to achieve biological control of rootknot nematodes by incorporating granules into soil around roots so that second-stage juveniles migrating from afar were trapped before they reach the root. Pot experiments carried out in field soil showed that such a strategy could be successful. When granules were applied at 4 and 8 g/L soil, the number of nematodes entering roots was reduced by 90 - 95%.