| Grade 0: | no disease; |
| Grade 1: | diseased spots on the root of blueberry seedlings less than 1.0 cm in size, but the plants were healthy; |
| Grade 2: | blueberry seedling root lesions of 1.0–2.0 cm, seedling leaves slightly withered, and lower leaves rarely shed; |
| Grade 3: | root lesions of blueberry seedlings were more than 2.0 cm, and seedling leaves were withered or fell off; |
| Grade 4: | blueberry seedling showed brown root rot, or the whole plant withered; |
| Grade 5: | blueberry seedling was dead. |
Fig. 1.
Inhibitory activity of strain TM11 against Fusarium commune and Fusarium oxysporum.
CK is F. commune and F. oxysporum alone; on the left side of each plate is the TM11 colony, and on the right side is the colony of F. commune or F. oxysporum.
Table I
Percentage inhibition of the mycelial colonies of the blueberry root rot pathogens Fusarium commune and Fusarium oxysporum by TM11 compared with the untreated control (CK) assessed in dual culture plates after 5 days of incubation.
| Strain | Fusarium commune | Fusarium oxysporum | ||
|---|---|---|---|---|
| Treatment colony radius (cm) | Percentage inhibition (%) | Treatment colony radius (cm) | Percentage inhibition (%) | |
| CK | 5.42 ± 0.08a | n.d. | 5.53 ± 0.05a | n.d. |
| TM11 | 1.73 ± 0.09b | 68.01 ± 1.45 | 1.90 ± 0.06b | 65.65 ± 1.22 |
[i] Data are presented as the mean ± SE. Data with different lowercase letters are significantly different at the 0.05 level.
Fig. 2.
Morphological characteristics of strain TM11.
A–B) colony of TM11 surface and base, C) conidiophores, D) conidia (scale 20 microns).
Fig. 3.
Phylogenetic trees based on ITS (A) and SSU (B) sequences using the neighbor-joining method.
The outgroup was Schizophyllum commune and Nectria berolinensis; numerical values above the branches are bootstrap percentiles from 1,000 replicates. Parsimony bootstrap values of more than 50% are shown at the nodes.
Fig. 4.
Inhibition of Fusarium oxysporum and Fusarium commune hyphal growth by Trichoderma asperellum strain TM11 under a microscope.
A) Strain TM11 causes lysis of F. oxysporum lysis of the hyphae (as shown by arrow), B) strain TM11 causes F. oxysporum lysis of the hyphae, and the cytoplasm has leaked out of that hyphal (as shown by arrow), C) strain TM11 to coil around the hyphae of F. commune (as shown by arrow), D) strain TM11 causes F. commune lysis of the hyphae, and the cytoplasm has leaked out of that hyphal (as shown by arrow).
Fig. 5.
Inhibitory effects of volatile metabolites and fermentation metabolites of Trichoderma asperellum strain TM11 on the growth of Fusarium commune and Fusarum oxysporum.
CK1 and CK2 represent the growth of F. commune and F. oxysporum on PDA; volatile metabolites mean volatile organic compounds (VOCs) and fermentation metabolites mean secondary metabolites, including alkaloids, ketones, esters, phenols and organic acids, such as erucamide, dibutyl phthalate and benzophenone.
Table II
Inhibitory effects of volatile and fermentation metabolites of Trichoderma asperellum strain TM11 on the growth of Fusarium commune and Fusarium oxysporum.
| Treatment | Fusarium commune | Fusarium oxysporum | ||
|---|---|---|---|---|
| Diameter of pathogen colony (cm) | Percentage inhibition (%) | Diameter of pathogen colony (cm) | Percentage inhibition (%) | |
| CK1 | 5.77 ± 0.05a | n.d. | 5.97 ± 0.05a | n.d. |
| Volatile metabolites | 3.83 ± 0.04b | 33.53 ± 0.72a | 3.77 ± 0.17b | 36.87 ± 2.74a |
| CK2 | 7.00 ± 0.22a | n.d. | 8.87 ± 0.05a | n.d. |
| Fermentation metabolites | 5.83 ± 0.05b | 16.67 ± 2.81b | 5.60 ± 0.08b | 36.84 ± 0.98a |
[i] Data are presented as the mean ± SE. Data with different lowercase letters are significantly different at the 0.05 level.
Table III
Types and relative abundances of antimicrobial metabolites produced in culture filtrates by Trichoderma asperellum strain TM11 based on HPLC–MS analysis of methanol extracts of fermentation broth.
| Chemical compound | Peak area | Retention time (min) | m/z |
|---|---|---|---|
| Erucamide | 221862.78 | 7.22 | 338 |
| Dibutyl phthalate | 158594.53 | 6.73 | 279 |
| Benzophenone | 24466.55 | 6.00 | 183 |
| Benzothiazole | 49711.09 | 7.09 | 136 |
| Citric acid | 49252.02 | 1.11 | 193 |
| Betainel | 6165.18 | 0.89 | 118 |
| Dipropyl phthalate | 5286.64 | 6.07 | 251 |
| alpha-Curcumene | 3619.38 | 5.43 | 203 |
| Phenylalanine | 2535.44 | 2.52 | 166 |
| 3-Hydroxycinnamic acid | 2500.47 | 2.90 | 165 |
| Chlorogenic acid | 1903.63 | 3.02 | 355 |
| 4-Hydroxybenzoic acid | 517.80 | 3.16 | 139 |
[i] Peak area indicates relative abundance.
Fig. 6.
Effect of blueberry seedling growth inoculated with Fusarium oxysporum or Fusarium commune.
A) Control group: inoculated with F. commune alone, B) treatment 1: F. commune inoculated first, and TM11 inoculated 48 hours later, C) treatment 2: TM11 inoculated first, then F. commune inoculated 48 hours later, D) control group: inoculated with F. oxysporum alone, E) treatment 1: F. oxysporum inoculated first, and TM11 inoculated 48 hours later, F) treatment 2: TM11 inoculated first, then F. oxysporum inoculated 48 hours later.
Table IV
Relative control effect of Trichoderma asperellum strain TM11 against Fusarium commune and Fusarium oxysporum disease in blueberry plants in a pot test after four weeks.
| Treatment | Fusarium commune | Fusarium oxysporum | ||
|---|---|---|---|---|
| Disease index | Percentage disease (%) | Disease index | Percentage disease (%) | |
| Control | 96.00 ± 3.27a | n.d. | 97.33 ± 1.88a | n.d. |
| Inoculation with TM11 first | 6.67 ± 1.89c | 93.05 ± 1.99a | 14.67 ± 1.89c | 84.76 ± 1.57a |
| Inoculation with pathogens first | 28.00 ± 3.27b | 70.92 ± 2.41b | 36.00 ± 3.27b | 62.46 ± 1.28b |
[i] Data are presented as the mean ± SE. Data with different lowercase letters are significantly different at the 0.05 level.
Table V
Effects of inoculation with Trichoderma asperellum strain TM11 and Fusarium commune or Fusarium oxysporum on SOD, POD, and CAT activities in leaf and root tissue of blueberry seedlings.
| Treatment | Leaf | Root | ||||
|---|---|---|---|---|---|---|
| CAT (U/g fresh weight) | SOD (U/g fresh weight) | POD (U/g fresh weight) | CAT (U/g fresh weight) | SOD (U/g fresh weight) | POD (U/g fresh weight) | |
| CK | 128.14 ± 5.86b | 208.01 ± 4.05c | 142.08 ± 4.05d | 140.96 ± 3.30c | 406.44 ± 2.02b | 150.86 ± 3.60d |
| TM11 | 224.59 ± 12.19a | 446.95 ± 3.21a | 353.22 ± 3.37a | 279.22 ± 1.36a | 490.13 ± 1.77a | 363.61 ± 11.14a |
| TM11 + F. commune | 217.09 ± 7.41a | 352.75 ± 4.79b | 309.32 ± 6.20b | 228.79 ± 3.43b | 469.36 ± 4.60a | 313.66 ± 4.75b |
| TM11 + F. oxysporum | 213.37 ± 9.78a | 349.54 ± 4.29b | 205.23 ± 0.30c | 274.70 ± 1.65a | 490.39 ± 12.74a | 306.37 ± 9.38b |
[i] Data are presented as the mean ± SE. Data with different lowercase letters are significantly different at the 0.05 level.