Evolution and ecophysiology of the industrial producer Hypocrea jecorina (Anamorph Trichoderma reesei) and a new sympatric agamospecies related to it

Abstract

Background: Trichoderma reesei, a mitosporic green mould, was recognized during the WW II based on a single isolate from the Solomon Islands and since then used in industry for production of cellulases. It is believed to be an anamorph (asexual stage) of the common pantropical ascomycete Hypocrea jecorina.

Methodology/principal findings: We combined molecular evolutionary analysis and multiple methods of phenotype profiling in order to reveal the genetic relationship of T. reesei to H. jecorina. The resulting data show that the isolates which were previously identified as H. jecorina by means of morphophysiology and ITS1 and 2 (rRNA gene cluster) barcode in fact comprise several species: i) H. jecorina/T. reesei sensu stricto which contains most of the teleomorphs (sexual stages) found on dead wood and the wild-type strain of T. reesei QM 6a; ii) T. parareesei nom. prov., which contains all strains isolated as anamorphs from soil; iii) and two other hypothetical new species for which only one or two isolates are available. In silico tests for recombination and in vitro mating experiments revealed a history of sexual reproduction for H. jecorina and confirmed clonality for T. parareesei nom. prov. Isolates of both species were consistently found worldwide in pantropical climatic zone. Ecophysiological comparison of H. jecorina and T. parareesei nom. prov. revealed striking differences in carbon source utilization, conidiation intensity, photosensitivity and mycoparasitism, thus suggesting adaptation to different ecological niches with the high opportunistic potential for T. parareesei nom. prov.

Conclusions: Our data prove that T. reesei belongs to a holomorph H. jecorina and displays a history of worldwide gene flow. We also show that its nearest genetic neighbour--T. parareesei nom. prov., is a cryptic phylogenetic agamospecies which inhabits the same biogeographic zone. These two species thus provide a so far rare example of sympatric speciation within saprotrophic fungi, with divergent ecophysiological adaptations and reproductive strategies.

Figure 1. Structure of the las1 locus in H. jecorina/T. reesei.

Intron-exon structure of the las1 locus in H. jecorina/T. reesei and position of PCR primers as inferred for T. reesei QM 6a.

Figure 2. Molecular phypogeny of H. jecorina sensu lato.

Bayesian circular phylogram inferred from the concatenated dataset of tef1, cal1 and las1 phylogenetic markers. Symbols at nodes correspond to posterior probabilities (PP) >95%. Filled circles correspond to PP in the concatenated tree, open stars, squares and polygons to PP in las1, cal1 and tef1 gene trees, respectively. The corresponding phylograms are given in Figure S1. The color code indicates the geographic region from which the isolates were obtained, as explained in the right top inset.

Figure 3. The mating type loci of H. jecorina.

(A) Schematic presentation of the mating type loci MAT1-1 and MAT1-2 and their flanking regions based on the H. jecorina data . Primers used to amplify the complete MAT-loci are indicated by gray arrows and primers for fragments of the mating type genes (Table 4) by black arrows. Numbers correspond to the respective proteins IDs in the T. reeesei genome database. (B) Restriction fragment patterns of the mating type loci, amplified with primers aF and aR (Table 4) and digested with PstI. MM, molecular marker (GeneRuler 1 kb ladder, Fermentas). The strains and their respective mating types are indicated as C.P.K. numbers. Small colored arrows show either present (filled) or absent (open) bands in RFLP profiles of C.P.K. strains in respect to the reference strains of H. jecorina for MAT1-1 and MAT1-2 .

Figure 4. Recombination analysis of H. jecorina and T. parareesei nom. prov.

Reconstruction of possible recombination networks build using the split decomposition method applied to the concatenated dataset (tef1 + cal1 + chi18-5). Upper shape: H. jecorina, low shape: T. parareesei nom. prov. Open and filled symbols at OTUs indicate MAT1-1 and MAT1-2 mating types respectively. Gaps were treated as missing characters throughout. All networks have been calibrated to fit one scale. The color scheme shows geographic origin of the strain as indicated in Fig. 2. Results from the PHT and Phi tests are shown by arrows and the respective P values, ‘rec +’ specifying positive recombination result and ‘rec -’specifying no recombination detected. PHT indicates the result of partition homogeneity test, Phi corresponds to results of Phi test. Double ended arrow lines show successful (solid line) and failed (dashed line) crossings.

Figure 5. Carbon source utilization by H. jecorina, T. parareesei nom. prov. and production of extracellular cellulases.

(A) Results of the single linkage cluster analysis (Pearson distance) applied to strains and based on growth on 95 carbon sources and water (Biolog FF MicroPlate ™) inferred from optical density values at 750 nm after 48 hours of incubation (linear growth stage) under ambient illumination conditions. (B) Volumetric cellulase activity of H. jecorina and T. parareesei nom. prov. Bars correspond to the average values per species and control strains with standard deviations (vertical lines), circles show the values obtained for individual strains. Control corresponds to cellulase overproducing and cellulase negative mutant strains QM 9414 and QM 9978 respectively, both derived from T. reesei QM 6a.

Figure 6. Photosensitivity map of H. jecorina and T. parareesei nom. prov.

Photosensitivity map of H. jecorina and T. parareesei nom. prov. constructed based on the two way joining cluster analysis. Framed squares show growth under conditions of sun light: white, black and grey frames correspond to photoinhibition, photostimulation and neutral photoresponse respectively. Bold font used for carbon sources indicates those which supported conidiation of H. jecorina (left list) and T. parareesei nom. prov. (right list) respectively.

Figure 7. Mycoparasitic ability of H. jecorina and T. parareesei nom. prov.

Results of dual confrontation tests between Trichoderma strains (inoculated on the left side) and the plant pathogenic fungi (inoculated on the right side): Sc–Sclerotinia sclerotiorum, FOX–Fusarium oxysporum complex, Fx–F. xylarioides, Alt–Alternaria alternata, Bot–Botrytis cinerea. Roman numbers indicate the weak (I), moderate (II), strong (III) and very strong (IV) ability of Trichoderma to inhibit the growth of the prey fungus. The ability to overgrow the mycelium of prey fungi is given in Arabic numbers on the similar scale. Antagonistic potential is calculated as the mean value for a strain to combat all five pathogens. The dashed lines correspond to the center position between confronted fungi.