title: Development of a soil granule based on the entomopathogenic fungus Metarhizium brunneum to control Agriotes spp.
Creators: name: Seib, Tanja Sarah Vanessa Michele
Publisher: name: UNSPECIFIED
Year: 2024

description: Crop protection faces particularly great challenges for the food production of a growing world population and in view of climate change. However, agriculture always has to contend with crop losses caused by plant pests and diseases. Pest insects of particular importance are wireworms, the larvae of click beetles, which cause reduced crop quality or crop losses in many cultured plants, such as potatoes, maize, cereals, asparagus or lettuce, by feeding on roots, tubers and seedlings. In Europe, larvae of the genus Agriotes, including the species Agriotes lineatus, Agriotes obscurus and Agriotes sputator, are considered a particularly severe problem. Because of their development in the soil, lasting two to five years, depending on the species and environmental conditions, they are difficult to control. Biological control strategies are advantageous, because, unlike chemical ones, they usually pose a low risk to the environment, applicators and consumers. Especially the effect of entomopathogenic fungi (EPF) against wireworms has been well documented. For these reasons, the aim of this dissertation was to develop a soil granule based on EPF to control larvae of the species A. lineatus, A. obscurus and A. sputator. 
Four steps were taken to achieve this goal. In the first step, an effective fungal strain against Agriotes larvae was determined. Then, the producibility of the fungal strain in liquid medium was optimized and the ability to formulate it by using a fluid bed dryer was investigated. In the fourth step, the influence of the production temperature on different quality factors of the developed granule as well as on virulence factors of the fungal strain were investigated.
To identify an effective fungal strain against the three Agriotes species, the virulence of six Metarhizium spp. and two Cordyceps spp. strains was compared (Chapter I). The Cordyceps strains proved to have no pathogenic effect on the larvae, whereas all Metarhizium strains were able to infect and kill the larvae and led to fungal growth on the cadaver. However, the effect of the individual Metarhizium strains on the three Agriotes species differed. Treatment with the Metarhizium brunneum strain JKI-BI-1450 resulted in the highest mortality against larvae of A. lineatus with 58% and A. obscurus with 94% and intermediate mortality of A. sputator with 56%, compared to the other fungal strains. The highest mortal effect against A. sputator larvae was caused by the Metarhizium robertsii strain JKI-BI-1442 with 94%. In order to explain the different mortality, the speed of germination, the germination after 96 h and the conidial size of the Metarhizium strains were determined. Conidia of the three M. brunneum strains (JKI-BI-1339, JKI-BI-1450 and LRC112) germinated between 96.8-98.5% after 96 h, whereas the conidia of the M. robertsii strains (JKI-BI-1441, JKI-BI-1442 and JKI-BI-1448) only germinated between 90.7-93%. τ, the time point where 50% of the maximal germinated conidia are germinated, was reached significantly faster by JKI-BI-1339 and JKI-BI-1450 after 14.5 h and 15.1 h, respectively. The longest conidia were formed by LRC112 and the widest by JKI-BI-1442. Subsequently, it was investigated whether these factors correlate with final mortality; however, this was not the case. Furthermore, it was investigated whether the effect of the fungal strains varies not only between the different Agriotes species, but also within larvae of one species between various populations obtained from different locations. Therefore, the effect of the two M. brunneum strains JKI-BI-1450 and ART2825 on different larval populations was investigated, as these fungal strains are already proven to be highly effective against Agriotes larvae. The mortality and mycosis effect of the two investigated fungal strains on the larvae of A. obscurus differed between the populations. The final mortality of A. obscurus larvae lay between 44-94% when treated with JKI-BI-1450 and between 30-95.6% when treated with ART2825. There was no difference observed between the populations of A. lineatus larvae and the final mortality was 38% for the JKI-BI-1450 treatments and 48% and 53.5% for the ART2825 treatments. Although the final mortality did not differ significantly between the different populations of A. sputator larvae and lay between 26-36% for JKI-BI-1450 and between 10.7-41.8% for ART2528, there were significant differences found in the time to larval death. Restricted mean time lost (RMTL) differed significantly between the populations and was between 3.6-18.5 for JKI-BI-1450 and between 6-21.5 for ART2825. The M. brunneum strain JKI-BI-1450 turned out to be a promising candidate for wireworm control, but the different efficacy against the Agriotes species and, in the case of A. obscurus, also against different populations, is problematic because so its field efficacy is difficult to predict. The development of a control strategy based on several fungal strains would be a possible solution.
In order to determine the producibility of the fungi, in chapter II all six Metarhizium strains were cultivated in three standard liquid media and the formation of submerged spores was evaluated. All fungal strains could be proliferated in liquid cultures and achieved spore concentrations of around 10^7 submerged spores ml^-1 except for LRC112 with a maximum of 6 × 10^4 submerged spores ml^-1. All further experiments were exclusively carried out with JKI-BI-1450 based on its efficacy. The submerged spore and biomass formation of JKI-BI-1450 was studied at different temperatures and after different incubation periods. The highest submerged spore formation was achieved after 48 h at 25 °C with 6.5 × 10^5 submerged spores ml^-1, as was the highest biomass formation also after 48 h, but at 30 °C with 3.2%. Afterwards, the growth of JKI-BI-1450 in a 3-Liter bioreactor at 25 °C was studied. It was found that submerged spore formation had its optimum between 42.5-48 h after inoculation with spore concentrations between 1.43-2 × 10^6 submerged spores ml^-1, whereas the biomass formation increased to 1.43% throughout 72 h duration of fermentation. To increase fungal production in liquid culture, the influence of the concentration of the media components (glucose, corn steep solid, sodium chloride) was evaluated. The increase of glucose and corn steep solid in the medium resulted in a steady rise of biomass production up to 28.0 g l^-1 and 23.0 g l^-1, respectively. In contrast, changing the sodium chloride concentration in the medium produced inconsistent results, although the highest concentration resulted in the greatest biomass formation with 14.7 g l^-1. 
After production in liquid culture, the fungal broth must be formulated to make it suitable for storage and application. In chapter III, fluid-bed drying was chosen for the formulation of JKI-BI-1450, in which a thin layer of fungal biomass was sprayed on millet and simultaneously dried. This formulation method proved to be well suited for the production of granule, requiring 4.5 mg of fungal biomass per gram of granule to achieve a granule colonization of nearly 100%. To increase or accelerate the fungal growth on the granule, chitin and various sugars were added to the fungal biomass before spraying and drying. The supplementation of chitin showed no positive effect, whereas the addition of the sugars led to an acceleration of colonization as well as of the formation of conidia, with the best results obtained for fructose. The addition of fructose increased granule colonization from 5.6 to 62.2% after one day of incubation and conidia formation from 3.35 × 10^5 to 1.13 × 10^6 conidia per granule grain after 5 days of incubation. The storage stability of the granule was also investigated, as this is important to provide sufficient shelf-life of the final product. After 12 weeks of storage at 5 °C, the granule colonization was still over 90%. The examination of granule quality under simulated field conditions showed that at the optimum residual soil moisture contents of 30% and 45%, the highest colonization was found at 25 °C and 30 °C with 98.9-100% and the most conidia per granule grain at 25 °C with 3.0-3.5 × 10^6. Furthermore, it was demonstrated that the reduction of colonization and formation of conidia was lower when only one environmental parameter (temperature and humidity) was unfavourable compared to both factors being disadvantageous. In addition, a fungicidal seed treatment (Moncut®) was identified as compatible with the developed granule without significant loss of granule colonization.
In chapter IV, it was investigated how the production temperature of the fungus affects granule colonization and conidia formation on the granule, as well as on germination, conidial size and virulence of the conidia. For this purpose, the biomass required for granule preparation was produced at different temperatures and the produced granules were also incubated at different temperatures. The fungus on the granules demonstrated the highest colonization with 87.17% and conidia formation with 2.77 × 10^7 conidia per granule grain when the biomass was produced at 25 °C. Furthermore, conidia were produced at different temperatures and then also incubated at different temperatures. Afterwards, their size, germination and virulence were investigated. The conidia produced at 20 °C were significantly wider with 2.21 µm compared to those produced at 25 °C and 30 °C with 2.19 µm. Furthermore, conidia produced at 15 °C reached τ significantly later (15.42 h) than conidia produced at 30 °C (12.31 h). The production temperature of the conidia had no effect on the virulence against Galleria mellonella larvae. The mortality ranged between 95.63-100% for all production temperatures with a survival time of 50% (ST50) between 3.97-4.54 days. With regard to the incubation temperature, it was found that at 30 °C smaller conidia were formed with 6.54 µm, germination was faster with τ of 11.40 h and the fungal treatment led to a faster mortality with a ST50 of 3.16 days. 
In summary, this dissertation identified M. brunneum JKI-BI-1450 as pathogenic against larvae of all three Agriotes species examined. This fungal strain can be produced in liquid medium and formulated into granule by fluid-bed drying. Both, production and formulation were improved by adjusting the process parameters or media composition and supplementing additives. Furthermore, it was shown that the quality of a fungus-based soil granule can already be influenced by the production conditions of the biomass used for it.