## 3 Results
### Metahizium Genotyping  
SSR-based genotyping and subsequent 5\({}^{\prime}\)-TEF-1\(\alpha\) sequencing of 653 _Metahizium_ isolates revealed the presence of 31 multilocus genotypes (MLGs) in addition to the applied production strain (Figure 4). The MLGs represented three species, i.e., _M. brunneumum_, _M. robertsii_, and _M. lepidiote_ (Table 2; Figure S1).  
\begin{table}
\begin{tabular}{l c c c c c c c c c c} \hline \hline  & \multicolumn{3}{c}{**BIPESCO 5**} & \multicolumn{3}{c}{_M. Brunneumum_} & \multicolumn{3}{c}{_M. Robertsii_} & \multicolumn{3}{c}{_M. Lepidiote_} \\ \hline
**Origin** & **Year** & **N** & **N** & **MLG** & **SG** & **N** & **MLG** & **SG** & **N** & **MLG** & **SG** \\ \hline Styria 1 & 2016 & 0 & 25 & 1 & 0 & 47 & 7 & 2 & 2 & 2 & 2 \\ Styria 1 & 2017 & 15 & 10 & 1 & 0 & 34 & 8 & 3 & 13 & 8 & 5 \\ Styria 1 & 2018 & 0 & 8 & 2 & 0 & 16 & 3 & 2 & 1 & 1 & 1 \\ Styria 2 & 2016 & 11 & 28 & 2 & 0 & 33 & 4 & 0 & 0 & 0 & 0 \\ Styria 2 & 2017 & 24 & 29 & 2 & 0 & 19 & 4 & 3 & 0 & 0 & 0 \\ Styria 2 & 2018 & 12 & 3 & 1 & 0 & 11 & 1 & 1 & 0 & 0 & 0 \\ Tyrol & 2016 & 20 & 130 & 5 & 3 & 5 & 1 & 0 & 0 & 0 & 0 \\ Tyrol & 2017 & 101 & 42 & 5 & 3 & 1 & 1 & 1 & 0 & 0 & 0 \\ Tyrol & 2018 & 30 & 2 & 2 & 2 & 0 & 0 & 0 & 0 & 0 & 0 \\ \hline \hline \end{tabular} N total number of isolates; MLG number of unique multilocus genotypes; SG MLG with a single isolate.  
\end{table}
Table 2: Numbers of isolates and MLG identified as _M. brunneum_, _M. robertsii_, or _M. lepidiote_ from the treated field sites. The applied strain BIPESCO 5 is shown separated from _M. brunneum_.  
Figure 4: Minimum spanning network (MSN) showing the relationship between the SSR genotypes isolated from the treated field sites (Styria 1, Styria 2, and Tyrol). Circle sizes are proportional to the number of isolates belonging to one MLG, the thickness of the line is proportional to genetic SSR based similarity of genotypes. MLG 24 represents the genotype of the applied strain BIPESCO 5.  
The MLG of the applied strain _M. brunneum_ BIPESCO 5 was detected in 213 isolates (32.6%) and at least once in every treated field site after application of the product. The MLG composition without the applied strain was significantly different (PERMANOVA, \(p\) < 0.001) among the three locations (Figure 5). _M. brunneum_ and _M. robertsii_ were isolated from all field sites. _M. robertsii_ was the dominant (54.3% in Styria 1, 67% in Styria 2) and, for Styria 2, genetically most diverse species in both Styrian trial sites, excluding the applied strain. Field site Styria 1 contained the highest diversity of MLGs from the three analyzed locations. Fifty-three percent of all MLGs were only found there. It was also the only field site where the species _M. lepidieta_ occurred (37.5% of the genotypes; but only corresponding to 9.9% of all isolates--the individual MLGs were usually found once). In Tyrol, _M. brunneum_ was the dominating species isolated from the soil (96.7% of samples without BIPESCO 5), exhibiting one major genotype (82.1% of all isolates). Two genotypes (MLG 7, MLG 31) were found in all three sampling sites. The majority (22) of the 31 MLGs was only isolated in Styria. Most of the MLGs (84%) were only found in one of the field sites. None of the genotypes was present at every sampling point of the different sites, but in both Styrian fields two genotypes were isolated at least 75% of the sampling times (Styria 1: MLG 31, MLG 32; Styria 2: MLG 15, MLG 32). In Tyrol, only one MLG was found in 75% of the samplings (MLG 23), but another genotype was found at least at 62.5% of the sampling times (MLG 16). Out of ten different _M. brunneum_ genotypes, only four clustered closely to the applied strain BIPESCO 5 (Figure 4; MLG 18, 20, 22, and 23). A maximum of two SSR loci differed in these genotypes by a maximum of four base pairs. The other, non-clustering _M. brunneum_ genotypes contained at least five variable loci (out of six).