B. cinerea isolation and pathogenicity tests
Thirty isolates of B. cinerea were obtained from tomato plants (10 isolates), vine plants (10 isolates), and strawberry plants (10 isolates). The survey was carried out over two successive years 2016 and 2017, in the north-central and south-eastern region of Algeria (Table S1; Supplementary data 1). Pathogenicity tests on half-apples revealed significant differences among the different isolates tested (Kruskal–Wallis test was done on diameters of the rot lesions, χ2 = 114.14, df = 29, P value < 0.05). The most virulent isolate of each culture was chosen for the biocontrol tests, from vine (BCV02), tomato (BCT04), and BCFr11 (strawberry) (Fig. 1).
Trichoderma identification and phylogenetic analysis
Fifteen isolates with macroscopic and microscopic characteristics of the genus Trichoderma were isolated from the different samples collected. Ten isolates were obtained from tomato; strawberries, vines rhizosphere and 5 isolates were obtained from Bio-compost®. Four genomic regions from all of these isolates were sequenced (Table S2; Supplementary data 1).
BLAST search of the sequences obtained during this work was performed and also, they were submitted to the ISTH TrichOKey (http://isth.info/tools/molkey/index.php) and TrichoBlast programs (http://isth.info/tools/blast/index.php). The results with the highest similarity percentages to the 15 sequences obtained in this study were selected for species identification.
Results revealed that the isolates TAS2, TAS4, TAS5, and TAS8 belong to Harzianum clade, and presented a (99%) of nucleotide identity with the reference sequences of the specie T. afroharzianum, for the tef1 (KP008850) and rpb2 genes (FJ442691) and (96%) with the species T. simmonsii, for acl1 gene (KJ665182). The isolate TBeC1 had a (99%) of nucleotide identity with the reference sequences of the species T. breve, of the clade Harzianum for the tef1 (KY688046) and rpb2 genes (KY687983) and (97%) with the species T. guizhouense for acl1 gene (KJ665030). The TLiC8 isolate present a (99%) of nucleotide identity with the reference sequences of the species T. lixii, of the clade Harzianum for the tef1 (FJ716622) and rpb2 and (98%) with the species T. atrobrunneum for acl1 gene (KJ664949). Using ITS sequences, they were identified as T. harzianum/H. lixii and showed (100%) of similarity to several species of the Harzianum clade.
Isolates TLS6, TLC2, and TLC4 showed (99%) of nucleotide identity to the reference sequences of T. longibrachiatum, for tef1 (JQ685867), rpb2 (JQ685883), and acl1 (KJ665057). However, for the ITS gene, they were identified as T. longibrachiatum and showed a percentage of (100%) nucleotide identity with several species belonging to the Longibrachiatum clade. The isolates TGS11 and TGS13 revealed (99%) of similarity to the reference sequences of T. gamsii, belonging to Viride clade based on the tef1 sequence (EF488134). In addition, the TGS7 and TGS10 isolates revealed (99%) of nucleotide identity with the reference sequence of T. gamsii for the acl1 gene (KJ665025). TAtC11 isolate revealed (99%) of similarity to the reference sequences of T. atroviride, clade Viride for tef1 (MH176994), rpb2 (FJ860518), and acl1 (KJ664952), while for the ITS gene, they were identified as species belonging to the clade Viride with (100%) nucleotide identity with several species of this clade. TBS1 showed (99%) of nucleotide identity with the reference sequences of T. brevicompactum species, for the tef1 sequence (EU338292, EU338283) and for ITS sequence, it was identified as T. brevicompactum and revealed (100%) nucleotide identity with several species of the Brevicompactum clade.
Phylogenetic trees were designed for each of the 4 gene regions studied, with the sequences of the 15 Algerian isolates and the reference sequences downloaded from GenBank. Thereby, the trees of the tef1, rpb2, and acl1 genes revealed the same phylogenetic distribution of the Algerian sequences obtained during this work and the presence of 4 distinct clades (Fig. 2) (Fig. S1, S2, S3; Supplementary data 2). The first one was the clade Harzianum, containing the isolates TAS2, TAS4, TAS5, TAS8, TBeC1, and TLiC8, the first 4 isolates were closely related to the reference strain of specie T. afroharzianum (G.J.S. 04-186), the TBeC1 isolate to the reference strain of specie T. breve (HMAS:248844) and the TLiC8 isolate to the reference strain of specie T. lixii (G.J.S. 97-96 = CBS 110080). The second was the Longibrachiatum clade, containing TLS6, TLC2, and TLC4 isolates, which were closely related to the reference strain of T. longibrachiatum (S328, CBS 816.68) for the 3 trees. The third clade was Viride, including TGS7, TGS10, TGS11, TGS13, and TAtC11 isolates; the isolates TGS7, TGS10, TGS11, and TGS13 are closely related to the reference strain of T. gamsii (GJS 04-09) and the TAtC11 isolate to the reference strains of T. atroviride (S360, CBS 142.95). The fourth was the clade Brevicompactum in which the isolate TBS1 belongs, and was closely linked to the reference strain of T. brevicompactum (CBS 109720 = G.J.S.04-381). However, the ITS tree revealed the same genetic distribution for clades, but the species were placed differently, thus making identification at the species level almost impossible (Fig. S1; Supplementary data 2).
In vitro antagonistic tests
Dual-culture technique
In the dual-culture test, the 15 isolates of Trichoderma inhibited the mycelial growth of the 3 most virulent isolates of B. cinerea as compared to the control without Trichoderma spp. with a range varying from 53 to 65% on PDA medium (Fig. 3). For each B. cinerea isolate, a significant Trichoderma effect was observed (ANOVA was performed on the percentage inhibition of mycelial growth, F = 82.33, df = 14,30, P < 0.0001 for BCV02, and F = 49.68, df = 14,30, P < 0.0001 for BCT04 and F = 49.29, df = 14,30, P < 0.0001 for BCFr11). The higher inhibition rate was recorded for T. gamsii (TGS7 isolate), T. atroviride (TAtC11 isolate) (Fig. S4; Supplementary data 3) and T. longibrachiatum (TAS8 isolate), which varied from 62 to 65%. T. longibrachiatum (TLC2 and TLC4 isolates) and T. breve (TBeC1 isolate) gave the lowest inhibition rates, ranging from 53 to 57%.
Effect of volatile compounds of Trichoderma on the mycelial growth of B. cinerea
Results revealed a significant difference in the effect of volatile substances produced by Trichoderma isolates on mycelial growth of the tested B. cinerea isolates (Kruskal–Wallis test was performed on the percentage inhibition of mycelial growth, χ2 = 43.018, df = 14, p value < 0.05 for BCFr11, χ2 = 42.477, df = 14, P value < 0.05 for BCV02 and χ2 = 43.262, df = 14, P value < 0.05 for BCT04) (Fig. 4). Volatile substances emitted by T. gamsii (TGS7 isolate) and T. atroviride (TAtC11 isolate) reduced mycelial growth of B. cinerea isolates by 64.49 and 62.31%, respectively, than the control (Fig. S5; Supplementary data 3). The lowest growth inhibition rate (18.41 and 19.72%) were reported for T. longibrachiatum (isolate TLS6) and T. afroharzianum (isolate TAS4), respectively.
Culture filtrates
A significant culture filtrates effect was observed for the 3 strains of B. cinerea (Kruskal–Wallis test was done on germling growth inhibition, χ2 = 33.407, df = 14, P value < 0.05 for BCFr11, χ2 = 41.267, df = 14, P value < 0.05 for BCV02 and χ2 = 53.082, df = 14, P value < 0.05 for BCT04) (Fig. 5). The best percentages of inhibition were recorded from the filtrates of T. brevicompactum (isolate TBS1) (90.68%) and T. atroviride (TAtC11 isolate) (68.72%), suggesting a high antifungal effect of these filtrates. While filtrates from the rest of Trichoderma spp. isolates revealed stimulation of germling growth for the 3 tested B. cinerea isolates, as compared to the control. Percentages of stimulation ranged from 4.25 to 46.31%, the highest percentage of stimulation being found for T. longibrachiatum (TLS6 and TLC2 isolates).
In situ test
The effect of spore suspension treatments of T. atroviride (TAtC11 isolate), T. brevicompactum (TBS1 isolate), and T. lixii (TLiC8 isolate) on the incidence of disease caused by B. cinerea (BCT04 isolate), in tomato cv. “KAWA” revealed significant differences. Biocontrol activity was observed for the 3 tested Trichoderma isolates for preventive and curative treatments. However, the highest percentages of disease control (DC) were recorded for T. brevicompactum (TBS1) with 64.43 ± 4.34% in preventive treatment and 51.35 ± 1.56% in curative treatment, while the lowest percentages of disease control (DC) were recorded for T. lixii (TLiC8) with 34.19 ± 4.54% in preventive treatment and only 28.46 ± 8.93% in curative treatment. Based on these results, T. brevicompactum (TBS1) was the most effective isolate for the control of grey mould on tomatoes caused by B. cinerea (BCT04) (Fig. 6).