LU132 and LU140 had different metabolic profiles

Phenotype microarrays have been used frequently for Trichoderma species and strain

identification and for mutant screening (Atanasova & Druzhinina, 2010; Bochner et al., 2001; Druzhinina et al., 2006; Friedl et al., 2008).

Applying this method, the metabolism and conidiation profiles of LU132 and LU140 could be compared and the two isolates could be well distinguished based on these profiles. The

biggest growth difference between LU132 and LU140 was found on α-D-glucose. After 43 h the OD750 of LU140 was significantly higher than in LU132 while both isolates still showed

the same catabolic activity. Phytagel, which is used as inoculating fluid for the Biolog FF plates, contains glucose as a carbon source. The presence of glucose in the Phytagel was unexpected and only discovered later. Glucose is known to induce carbon catabolite repression (CCR) in T. reesei and other fungi (Portnoy et al., 2011; Ronne, 1995). Because glucose is the favoured carbon source, it is metabolised first while the gene expression of other carbon metabolising enzymes is supressed. The fact that the catabolic activity on glucose as a single compound was similar in LU140 and LU132 but that LU140 grew better on glucose would suggest that the presence of glucose in all the other wells elevated the growth data obtained from LU140 in the phenotype microarray.

The biggest catabolic differences between LU132 and LU140 were found on two L-amino acids (L-asparagine and L-pyroglutamic acid) and on one peptide (L-alanyl-glycine). LU140 could catabolise these molecules more efficiently but both isolates grew similarly on them. When the effect that glucose had on the growth of LU140 was taken into account, LU132 resulted in more growth than LU140 on these nitrogen compounds. A high level of catabolism resulting in less growth in LU140 and a low level of catabolism resulting in more growth in LU132 indicate that these compounds may play roles in different metabolic pathways in LU140 and LU132.

The catabolism of, and growth on, the glucoside salicin was significantly increased in LU132, especially when the glucose effect on LU140 was taken into account. Salicin has been shown to induce low activity of β-glucosidase in Aspergillus niger (Unno et al., 1993). However, those authors found that the enzyme activity on salicin was very low (7%) in comparison to activity on the main substrate cellobiose (100%). Even though β-glucosidases have been found to play an important role in the cellulolytic system of T. atroviride and T. reesei

(Kovács et al., 2008) and in the mycoparasitic activity of T. atroviride (Lorito, Hayes, Dipietro, Woo & Harman, 1994), there was no information available about the relationship between salicin and β-glucosidases in Trichoderma. Cellobiose was also catabolised significantly more efficiently by LU132 than by LU140 but the difference was very small compared to salicin. This indicates that the metabolism of salicin is likely to be triggered by a different unknown enzymatic pathway while the cellulolytic systems of LU132 and LU140 are likely to be similar.

The compound N-acetyl-D-glucosamine (NAG) resulted in significantly more mycelial growth in LU140 than in LU132 after 43 h. However, when the growth difference that

glucose caused was taken into account, LU132 produced slightly more mycelium than LU140 (not significantly). Apart from that, LU140 still exhibited a higher catabolic activity on NAG than LU132. NAG is a nitrogen containing hexosamine, which is known to trigger the

expression of the gene nag1, which encodes N-acetyl-β-D-glucosaminidase 1 (Mach et al., 1999; Peterbauer et al., 1996). N-acetyl-β-D-glucosaminidases (glycoside hydrolase family 20) cleave chitobiose dimers into monomers and are involved in chitin degradation of fungal cell walls (Brunner et al., 2003), in mycoparasitism (Zeilinger et al., 1999) and mycelial growth on chitin (Lopez-Mondejar et al., 2009). The differential metabolism of NAG might therefore be responsible for the different growth and antagonism characteristics of LU132 and LU140.

The mycelial growth of the two isolates varied on single compounds but was very similar on average across all 96 wells on the Biolog FF plates. In contrast to that, the average conidiation between LU132 and LU140 was significantly different. LU140 conidiated faster and more intense on a number of compounds, but as observed in the initial colony morphology experiments, a high conidiation score did not coincide with a slow growth or vice versa. The review by Atanasova & Druzhinina (2010) describes the application and methodology of phenotype microarrays for Trichoderma mutant characterisation. The authors noted that there might be inconsistencies with the absorbance measurement at 490 nm in imperfect fungi as the colour formation did not correspond with growth. As a result of this, a number of studies only analysed the biomass at OD750 and ignored the other data (Druzhinina et al., 2006;

Komon-Zelazowska et al., 2007; Seidl et al., 2006; Tanzer et al., 2003). The present study shows however, that the average absorbance data (average of all wells) at 490 and at 750 nm correlated very well and were well reproducible. While studying the metabolic profiles of fungal communities (Di Lonardo et al., 2013) the authors came to the same conclusion and even noted that the OD490 data lead to greater statistical discrimination between species. The

discrepancies between the two absorbance readings on individual compounds consequently supply very interesting information about the catabolism and utilisation of specific