Control Effect and Mechanism of Trichoderma asperellum TM11 against Blueberry Root Rot

Plant pathogens: Blueberry root rot (F. oxysporum and F. commune), blueberry branch blight (Pestalotiopsis clavispora), and blueberry brown leaf spot (Alternaria alternata) were obtained from the Forestry College, Guizhou University. Sorghum anthracnose (Colletotrichum sublineola), corn stalk rot (Fusarium verticillioides), pepper anthracnose (Colletotrichum acutatum), and wheat head blight (Fusarium graminearum) were obtained from the Northwest A&F University College of Plant Protection (China). These fungal species were stored in a refrigerator at −80°C at the Pathology Laboratory, Forestry College, Guizhou University (China).

Soil samples: Rhizosphere soil samples were obtained in April 2019 from organic blueberry fields (107°43′07″E, 26°25′49″N) in Majiang County, Guizhou Province, China. Five sample plots (5 m × 5 m) were set along a contour line near the blueberry root rot's incidence area. Eight healthy blueberry plants were randomly selected from each quadrat and dug up. Their roots were obtained, the excess soil particles attached to the roots were removed, the rhizosphere soil was retained, and rhizosphere soil samples were collected by shaking the roots. The soil samples were put into sterile bags, brought back to the laboratory in a portable low-temperature incubator, and stored in a refrigerator at 4°C to isolate potential biocontrol fungi.

Biocontrol fungi were isolated by the dilution-plate method (El Komy et al. 2015). Equal amounts of each of the soil from the five plots were mixed, and a 1 g sample was placed in a conical bottle. Then, 20 ml of sterile water was added, and the mixture was shaken for 1 min to disperse the soil into a soil suspension evenly. After standing for 10 min, 200 μl aliquots of the supernatant were spread-plated on the surface of rose bengal agar plates (5 g/l peptone, 20 g/l dextrose, 1 g/l dipotassium hydrogen phosphate, 0.5 g/l magnesium sulfate, 0.01 g/l rose bengal dye, 0.1 g/l chloramphenicol, and 15 g/l agar). Ten replicate plates were established. The plates were cultured at 28°C for 3–5 days in darkness, and a single colony hyphal tip of suspected Trichoderma was selected from the culture medium, transferred to PDA (potato broth from 200 g/l, 20 g/l dextrose, and 15 g/l agar), cultured at 28°C and purified 3–5 times. The PDA plate contained only a single colony with the same characteristics. The biocontrol fungi with antibacterial effects against F. oxysporum and F. commune were screened by the plate antagonism method.

Under sterile conditions, a 5-mm-diameter hyphal plug of biocontrol fungi cultured on PDA and the same-sized hyphal plug of F. oxysporum or F. commune were placed 2 cm away from the periphery of a Petri plate containing PDA (90 mm diameter) on opposite sides of the plate for both F. oxysporum and F. commune. Control plates consisted of a mycelial disc from the pathogen placed alone on PDA plates. The experiment included 3 replicates, and the plates were cultured at 28°C for 5 days in darkness. The inhibition of mycelial growth of the pathogen was then calculated as a percentage using the formula below (da Silva et al. 2016): Inhibition (%)=100%×(1−AB) Inhibition\;\left( \% \right) = 100\% \times \left( {1 - {A \over B}} \right) where A and B are the colony diameters of plant pathogens in the control and dual culture plates, respectively. Then, the broad-spectrum antimicrobial activity and chitinase enzyme production capacity of the selected biocontrol fungi were measured by the plate antagonism method and plate transparent circle method (0.01 g Fe₂SO₄ · 7H₂O, 0.7 g K₂HPO₄, 0.3 g KH₂PO₄, 0.5 g MgSO₄ · 7H₂O, 20 g agar, 250 ml 1% colloidal chitin, 750 ml distilled water, pH 7.0–7.2).

Finally, a biocontrol fungus with broad-spectrum antimicrobial activity and chitinase enzyme production capacity was identified by morphological and molecular biological methods. The colony characteristics (colony color, colony shape, and edge characteristics) were observed and photographed with a camera. The morphology of hyphae and spores of the biocontrol fungus were observed using a microscope, and morphological identification was completed with reference to Nguyen et al. (2018). The DNA of biocontrol fungi was extracted from mycelium using a Fungal DNA Mini Kit (Soleibao Biotechnology Co., Ltd., China). The ITS region of the biocontrol fungal strain was amplified using the universal primers ITS (5′-TCCGTAGGTGAACCTGCGG-3′, 5′-TCCTCCGCTTATTGATATGC-3′) and SSU (5′-GTAGTCATATGCTTGTCTC-3′, 5′-CTTCCGTCAATTCCTTTAAG-3′), and polymerase chain reaction (PCR) was performed using the following cycle conditions: initial denaturation at 98°C for 3 min; followed by 36 cycles of denaturation at 98°C for 10 s, annealing at 56°C for 10 s, extension at 72°C for 10 s and a final extension for 5 min at 72°C; and storage at 12°C. The resulting 587 bp PCR products were assayed by 1.2% agarose gel electrophoresis. The PCR product of the biocontrol fungus was sent to Qingke Xingye Biotechnology Co. Ltd. (China) for sequencing in both the forward and reverse directions, and these were then used to produce a consensus sequence. The sequence of the obtained biocontrol fungus was analyzed by the BLAST program of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). The ITS and SSU sequences were compared using nucleotide BLAST with default settings and megablast (highly similar sequences) as the selected program.

The hyperparasitism effect of the TM11 strain on the hyphae of F. commune or F. oxysporum was determined by confrontation culture on a glass slide. First, the slide was soaked in absolute ethanol for 10 min, the ethanol was removed, and the slide was dried naturally. Then, the slide was placed in an empty Petri dish, and a thin layer of PDA was poured on the slide. After allowing to set, the TM11 and F. oxysporum or F. commune mycelia were 3 cm apart of the slide to form a two-point confrontation and cultured at 28°C for 3 days. Once the two strains intersected, they were observed and photographed under a light microscope (Haran et al. 1996).

The inhibition of F. oxysporum and F. commune by volatile metabolites produced by strain TM11 was determined by sandwich plate. Under sterile conditions, strain TM11 and F. oxysporum and F. commune plugs of the same size (diameter = 5 mm) were obtained and inoculated in the center of two PDA plates. The F. oxysporum and F. commune inoculated plate were placed inversely over the TM11 plate to form a double dish set and cultured for 5 days at 28°C. Controls consisted of a PDA plate inoculated with F. oxysporum or F. commune and the uninoculated PDA. Three replicates were set up for each treatment, arranged in a completely randomized design, and cultured for 5 days at 28°C. The inhibition of mycelial growth of the pathogen was then calculated as a percentage using the formula previously described.

Inhibitory effects of the fermentation broth of strain TM11: A 5-mm-diameter hyphal plug of strain TM11 was inoculated into a 250 ml triangular bottle containing 100 ml potato dextrose broth (PDB; potato broth from 200 g/l and 20 g/l dextrose) and cultured for 7 days at 28°C and 160 r/min in the dark. The fermentation broth was filtered through four layers of sterile gauze, and a nylon water system 0.22 μm (Ø 25 mm) disposable filter was used to remove the mycelium and spores. Then, the resulting filtrate was mixed with PDA (adding more agar to the PDA to ensure it was set) at a ratio of ½ to make an inhibition plate. Mycelial colonized agar plugs of F. oxysporum or F. commune with a diameter of 7 mm were cut and inoculated in the center of a TM11 broth-amended PDA plate. Control plates consisted of F. oxysporum or F. commune inoculated on unamended PDA plates. Three replicates were set up for each treatment, and the plates were arranged in a randomized design and incubated for 5 days in a constant temperature environment of 28°C. The colony diameter was measured, and inhibition was calculated (El Komy et al. 2015). The data were statistically analyzed using the equation previously described.

A 5-mm-diameter hyphal plug of strain TM11 cultured on PDA was used to inoculate a 250 ml triangular bottle containing 100 ml PDB and cultured for 7 days at 28°C and 160 r/min. The resulting culture was then centrifuged at 12,000 × g and 4°C to obtain the supernatant, which was subjected to metabolite extraction with 50% methanol buffer. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was used to detect the fermentation extract metabolites and their relative concentrations. Detection conditions of HPLC-MS: TripleTOF^® 5600 (SCIEX, UK); chromatographic analysis column (Acquity UPLC HSS T3, 100 mm × 2.1 mm, 1.8 μm); chromatographic separation conditions: the column temperature was 35°C; the mobile phase was solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid); the flow rate was 0.4 ml/min; the sample injection volume was 4 μl; liquid elution gradient setting: 0–0.5 min, 5% B; 0.5–7 min, 5–100% B; 7–8 min, 100% B; 8–8.1 min, 100–5% B; 8.1–10 min, 5% B. Mass spectrometry conditions: ESI source, collected using ESI positive and negative modes, curtain gas (CUR) pressure was 30 psi, nebulizer gas (GS1) pressure was 60 psi, desolvation gas (GS2) pressure was 60 psi, and desolvation gas temperature was 650°C; in ESI positive mode, the atomization voltage (ISVF) was 5,000 V, and the declustering voltage was 100 V; in ESI negative mode, the atomization voltage (ISVF) was −4,500 V, and the declustering voltage was −100 V. Mass spectrometry data was collected in IDA mode; the TOF MS scanning range was 60–1,200 m/z; metabolite precursor ions were selected, and after collision-induced dissociation, two-dimensional mass spectrometry analysis was performed to detect their fragment ions.

Control effect of TM11 on blueberry potted plants: F. oxysporum and F. commune and strain TM11 were inoculated into separate 250 ml triangular bottles containing 100 ml PDB and shake-cultured for 7 days at 28°C and 160 r/min. Four layers of sterile gauze were used for filtration to remove mycelia, and the resulting spore suspensions were diluted with distilled water to dilute the broth containing the spores with a concentration of 1 × 10⁵ spores/ml. One-year-old healthy blueberry seedlings (‘Legacy’) were selected for testing. Four treatment groups were set up: 1) pathogen only control inoculated with 50 ml spore suspension of F. oxysporum or F. commune alone; 2) TM11 alone, 50 ml spore suspension of strain TM11; 3) TM11 prior to pathogen, 50 ml spore suspension of strain TM11 48 h prior to inoculation with 50 ml spore suspension of F. oxysporum or F. commune; 4) TM11 48 h after pathogen, 50 ml spore suspension of F. oxysporum or F. commune 48 h prior to inoculation with 50 ml spore suspension of TM11; 5) negative control, water. The growth and disease incidence based on leaf drop and root rot of blueberries were observed, the disease incidence and disease index were determined, and the incidence and relative control effects were calculated according to the following formulas (Elshahawy and El-Mohamedy 2019): A(%)=(BC)×100% A\left( \% \right) = \left( {{B \over C}} \right) \times 100\% D=(∑E×FC×G)×100% D = \left( {\sum {{{E \times F} \over {C \times G}}} } \right) \times 100\% H(%)=(D1−D2D1)×100% H\left( \% \right) = \left( {{{D{\it 1} - D{\it 2}} \over {D{\it 1}}}} \right) \times 100\% where A is disease incidence; B is the total number of diseased plants in each treatment; C is the total number of replicate plants in each treatment; D is disease index; E is the number of diseased plants at all levels; F is the number of corresponding grades; G is the value of he highest disease level; H is a percentage disease control effect; D1 is the control disease index, and D2 is the treatment disease index. Classification criteria of the disease index, which is divided into six levels according to the degree of disease occurrence:

The pot experiment was carried out in a greenhouse. Several nutrient bowls with One-year-old blueberry seedlings that were healthy, disease-free, and consistent in growth were selected, and 30 cm diameter pots were potted in sterilized humus soil and inoculated by root injury irrigation. A sterilized scalpel was inserted into the soil near the plant, causing root wounds. Each blueberry seedling was infused with 50 ml of spore suspension or, in the control group, an equal amount of sterile distilled water. No fertilization was applied during the growth period, watering was performed every three days, and the plants were harvested 30 days after the last inoculation and management. Growth medium prepared from organic humus, crushed pine needles, leaves, high-quality sawdust, imported mix of coconut coir, and rice bran and fermented humus soil was purchased from Luyuan Meijia Agricultural Technology Co. Ltd. and autoclaved at 121°C for 2 h before use to remove microorganisms from the substrate.

The plants in the experiment were used to assess the effects of strain TM11 on defense enzyme activity. SOD, POD and CAT were determined by the nitrogen blue tetrazole (NBT) reduction method, visible spectrophotometry, and UV spectrophotometry (Sundar et al. 2004; Guo et al. 2020). Tissue samples were harvested from each of three replicate plants.

A phylogenetic tree was constructed using the neighbor-joining method with MEGA7 (Kumar et al. 2016). The data were statistically analyzed by one-way analysis of variance (ANOVA) using IBM^® SPSS^® Statistics 25. Comparisons among means were performed using Duncan's multiple-range tests (p < 0.05).

As shown in Fig. 1, ten strains with the same colony characteristics were isolated from the blueberry rhizosphere soil samples, and we found that the growth rate of TM11 mycelia was faster than that of F. oxysporum and F. commune mycelia. TM11 quickly colonized the PDA plate and sporulated on the surface of the Fusarium colony, covering the hyphae and surrounding the pathogen colony in different forms (Fig. 1). TM11 inhibited F. oxysporum and F. commune by 65.7% and 68.0%, respectively (Table I). In addition, we also found that TM11 is resistant to P. clavispora, F. graminearum, C. sublineola, F. verticillioides, C. acutatum, and A. alternata (Fig. S1), and produces chitinase (Fig. S2). So TM11 was selected for subsequent experiments. The morphology of TM11 inoculated into PDA, colonies on PDA, sporulation structure, and spore morphology were observed. TM11 grew rapidly and filled the dish within three days of incubation. The hyphae grew radially, the colony edges were neat, nearly round, white to green in color, and fluffy, and the aerial hyphae were cotton-wool-like (Fig. 2A and 2B). The conidiophores were produced in paired branches on either side, often branched 2–3 times (Fig. 2C). The conidia were numerous and oval or round (Fig. 2D). The ITS and SSU sequences for TM11 obtained through BLAST revealed that TM11 was 99–100% identical to T. asperellum (GenBank accession No. MW766992 for ITS and OQ592847 for SSU). Neighbor-joining (NJ) phylogenetic trees were constructed based on ITS and SSU sequences (Fig. 3). The results showed that TM11 and sequences of T. asperellum clustered in the same clade and had the closest relationship, and TM11 was identified as T. asperellum based on combined morphological and molecular identification.

Fig. 1.

Fig. 2.

Fig. 3.

As shown in Fig. 4, after TM11 hyphae were in contact with F. oxysporum hyphae, the F. oxysporum hyphae showed obvious lysis (Fig. 4A and 4B), and F. commune hyphae were reduced and slowly disintegrated (Fig. 4C and 4D).

Fig. 4.

As shown in Fig. 5, both the volatile and fermentation metabolites of TM11 had different inhibitory effects on the growth of both F. oxysporum and F. commune. For F. oxysporum, the percentage inhibition was the same between volatiles and broth metabolites. However, there were differences for F. commune: volatile metabolites had a significant inhibitory effect on colony growth. In contrast, the fermentation metabolites had no significant inhibitory effect on colony growth, as shown in Table II. The volatile metabolites of strain TM11 inhibited F. commune and F. oxysporum by 33.5% and 36.9%, respectively; TM11 fermentation metabolites had the weakest inhibitory effect, with inhibition rates on F. commune and F. oxysporum of 16.7% and 36.8%, respectively (Table II).

Fig. 5.

Compounds in methanol extracts of the fermentation broth of T. asperellum strain TM11 were analyzed by HPLCMS. The results showed that under positive ion mode, the total ion current method was conducive to providing information about the compounds in the extract, so positive ion mode was used to analyze the antagonistic substances in the methanol extract (Fig. S3). Strain TM11 was shown to secrete a variety of antagonistic metabolites. The metabolite analysis results are shown in Table III. There were 12 chemical compounds identified, and their relative abundances (peak area) were as follows: erucamide > dibutyl phthalate > benzophenone > benzothiazole > citric acid > betainel > dipropyl phthalate > alpha-curcumene > phenylalanine > 3-hydroxycinnamic acid > dioctyl phthalate > chlorogenic acid > 4-hydroxybenzoic acid.

As shown in Fig. 6, in the negative control, inoculation with TM11 and F. commune or F. oxysporum significantly reduced disease compared with inoculation with only F. commune or F. oxysporum (Table IV) (Fig. 6B, 6C, 6E and 6F). The effect of the TM11 inoculation sequence on disease caused by F. commune or F. oxysporum significantly differed, with strain TM11 significantly reducing blueberry root rot when inoculated 48 h before inoculation with F. commune or F. oxysporum (treatment 1) (Fig. 6C and 6F) compared with inoculation with the pathogen 48 h before inoculation with TM11 (treatment 2) (Fig. 6B and 6E). As shown in Table IV, with the disease index in plants inoculated only with F. commune being 96.00, the disease index of treatment 1 was 6.67, which was 89.33 less than that of the pathogen control treatment, and the relative control effect was 93.05%. The control group disease index decreased by 68.00, and the relative control effect was 70.92%. The disease index in plants inoculated only with F. oxysporum was 97.33%. Treatment 1 inoculated with TM11 48 h before inoculation with F. oxysporum reduced the disease by 84.76%; treatment 2 inoculated with F. oxysporum first and inoculated with TM11 after 48 h reduced the disease by 62.46%. This indicated that TM11 had a strong inhibitory effect on blueberry root rot.

Fig. 6.

As shown in Table V, the CAT, SOD, and POD enzyme activities in the roots and leaves of blueberry seedlings inoculated with TM11 were significantly higher than those in plants inoculated with the water-negative control, and the root enzyme activity was higher than the leaf enzyme activity. Compared with the negative control, the activities of CAT, POD, and SOD in the roots inoculated with TM11 were increased by 1.01 times, 1.41 times and 0.21 times, and the activities of CAT and POD in the leaves were increased by 0.85 times, 1.49 times, and 1.15 times, respectively. For leaves, TM11 inoculation and inoculation of TM11 and F. commune or F. oxysporum on CAT enzyme activity. The activities of SOD and POD were the highest after inoculation with TM11 alone, which were 447.0 U/g and 353.2 U/g, respectively. There was no significant difference between TM11 inoculation and mixed inoculation of TM11 and F. commune or F. oxysporum on SOD activity for roots. The enzyme activities of CAT and POD were significantly higher in plants inoculated with TM11 alone, which were 279.2 U/g and 363.6 U/g, respectively.