Abstract
The application of Trichoderma spp. has the potential to reduce not only mineral fertilizer use in agriculture but also improve soil health through increased soil biological activity. Trichoderma spp. have shown potential as bio-control agents and plant growth promoting ability, but little attention has been paid to the effect of Trichoderma spp. inoculation on nutrient availability and the soil microbiome. In this study, we evaluated the effect of Trichoderma spp. inoculation on nitrogen mineralization and quantified soil enzymatic activities along with plant growth promotion potential. The influence of Trichoderma spp. and organic amendments on the soil fungal community was also investigated. For this purpose, pots and incubation experiment was carried out, and seven treatments were set as follows; poultry compost (PC), poultry compost + RW309 (PCT), cattle compost (CC), cattle compost + RW309 (CCT), rapeseed oil cake (OC), inorganic fertilizer (N) and only soil (S) were set as control. We evaluated that Trichoderma sp. RW309 produced indole-3-acetic acid, which suggested that it could contribute to plant growth enhancement during early plant growth. Inoculation of RW309 with organic materials stimulated nitrogen mineralization and increased soil phosphatase activity. Furthermore, RW309 altered the fungal community in rhizosphere soil. However, cattle compost was a more suitable culture medium for RW309 than poultry compost in terms of nitrogen mineralization, soil enzyme activity, and growth of RW309. In conclusion, Trichoderma sp. RW309 could be considered for use as a bioorganic fertilizer in combination with organic compost to minimize the use of mineral fertilizers.
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Introduction
Overuse of mineral fertilizers and mono-cropping agriculture reportedly deteriorate soil quality (Komatsuzaki and Ohta 2007). The continuous use of mineral fertilizers affects the long-term soil nutrient status, which ultimately affects the soil microbial community and their function (Kuzyakov et al. 2000; Xiong, 2016) and can have a negative environmental impact. Organic materials, including cattle and poultry manure, can improve soil biological, chemical and physical properties while also enhancing crop growth and reducing the incidence of plant diseases when compared with mineral fertilizer (Abiven et al. 2009; Annabi et al. 2011; Eden et al. 2017) as well as reduced nitrogen losses through leaching (Chalhoub et al. 2013).
Soil microorganisms play very important roles in ecological functions such as formation of soil aggregates, nutrient cycling, organic matter decomposition, plant growth promotion and mitigation of soil borne diseases (Zhang 2012). Microbial community structure and soil microbial activity are important indicators of soil quality because both factors are sensitive to variation in cropland management practices (Bending et al. 2002). Soil enzyme activities are sensitive to environmental change and have been suggested as a soil quality indicator because of their relationship with soil biology (Henry 2013) and their rapid reactions to changes in management and the soil environment (García-Ruiz 2009; Burke et al. 2011). Soil enzymes are closely engaged with soil nutrients (Gu 2019). Therefore, using organic materials to manage soil fertility and microbial activity could have potential to improve soil productivity and quality (Hartmann et al. 2015). Management strategies such as the use of organic fertilizers, and crop rotation have been shown to preserve the soil microbial community and their functions (Shen et al. 2014; Li 2016; Xiong et al. 2016).
Trichoderma spp. can be used as a supplement to reduce mineral fertilizer use and also functions as a biocontrol agent for soil borne diseases (Molla et al. 2012; Cai 2015). They grow rapidly and inhabit the rhizosphere, phyllosphere, plant seed and soil (Halifu et al. 2019). Trichoderma spp. promote plant growth and development (Bae 2011), possibly through production of auxins like primary and secondary metabolites such as indole-3-acetic acid (IAA; (Sofo 2011). Therefore, soil microorganisms and their activity in soil can have profound effects on nutrient uptake, soil health and plant growth (Bonfante and Anca 2009; Mendes 2011; Berendsen et al. 2012). However, little attention has been paid to the effect of Trichoderma spp. inoculation on nutrient availability and the soil microbiome. The aims of this study were to evaluate the plant growth promoting ability of Trichoderma spp. and to determine the mechanism for plant growth enhancement in terms of nitrogen (N) mineralization, release of enzymes and fungal traits for plant growth promotion. This study evaluated the effect of Trichoderma spp. on N mineralization, soil enzyme activities and plant growth promoting potential. The influence of Trichoderma spp. and organic amendments on the soil fungal community was also investigated.
Materials and methods
Soil chemical properties and organic materials
Gray lowland soil was collected from the Agriculture Research Station of the University of Yamanashi, Kofu, Yamanashi, Japan (N35°60′39.5, E138°57′82.9). The soil was collected from the upper layer (0–20 cm) and passed through a 2-mm sieve to remove the stones, root materials and surface litter. The soil pH and electrical conductivity were 6.79 and 0.11 mS cm−1, respectively. The soil contained 23.1 mg kg−1 nitrate–N (NO3−–N) and 190 mg kg−1 available phosphate.
Three different types of organic materials (poultry compost, cattle compost and rapeseed oil cake) were used in this study. Organic materials were air-dried, crushed into small particles through a grinder and passed through a 2-mm sieve for further analysis. Triplicate samples of poultry compost (PC), cattle compost (CC) and rapeseed oil cake (OC) were measured for total N and total carbon (C) on a NC analyzer (SUMIGRAPH® NCH-22F, Sumika Chemical Analysis Service, Ltd., Tokyo, Japan) by Win et al. (2020). Chemical properties of the organic materials are shown in Table 1.
Trichoderma sp. strain
Trichoderma sp. strain RW309, hereafter known as RW309, was isolated from rhizosphere soil of Glycine max grown in Yamanashi, Japan. This strain was most closely related to Trichoderma hamatum based on internal transcribed spacer region sequencing (Accession No. LC586215) and having an ability to inhibit the growth of plant pathogen (Sclerotium rolfsii) checked by confrontation assay (Fig. S1). The initial spore density was measured by the dilution plating technique to obtain 3.1 × 109 colony forming units (cfu) mL−1. A spore suspension of this strain was prepared according to Zhang (2013).
Plant growth promoting ability of RW309
A plant growth experiment was conducted to evaluate the effect of RW309 on plant growth and pots were filled with 300 g of air-dry soil and phosphate (150 mg kg−1 of soil). Brassica rapa var. perviridis (Brassica) and Lactuca sativa var. crispa (lettuce) were grown for 4 weeks with the following treatments: B. rapa with RW309 (Br + Tri), B. rapa alone (Br), lettuce with RW309 (Lc + Tri) and lettuce alone (Lc). 1 ml of the RW309 (Trichoderma hamatum) spore suspension was inoculated at the start of plant growth. Organic materials were not used in this experiment. Plants were grown in a growth chamber at 25 ºC under a 18 h light: 6 h dark (day/night) cycle for 4 weeks; the plants were then harvested and the biomass of shoot and root parts was determined. The experiment was conducted by triplicate.
Strain RW309 traits
Calcium phosphate solubilization by RW309 was investigated through the use of media described in Pikovskaya (1948) using Pikovskaya’s agar (HiMedia laboratories, Mumbai, India). RW309 was grown on this media in triplicate and incubated for 1 week at 25 °C. Clear zones around RW309 colonies were evidence of phosphate solubilization. Results were compiled through the following index: + detectable clear zone, − non-detectable clear zone, ± detectable clear zone but very weak.
Siderophore production was investigated through growth of RW309 on modified Chrome Azurol S (CAS) medium (Schwyn and Neilands 1987; Pérez-Miranda et al. 2007). RW309 was grown on PDA media (OXOID LTD, Basingstoke,Hampshire, England) in triplicate and incubated for 24 h, and then covered with 10 ml of media containing the following components: 7.3 mg of hexadecyl trimethyl ammonium bromide, 6.04 mg of CAS, 1 mL of 1 mM FeCl3·6H2O, and 3.04 g of piperazine-1,4-bis (2-ethanesulfonic acid). After pouring the CAS media then plates were incubated for 7 days at 25 °C. The color of the CAS agar changed from blue to light yellow or orange. Color changes were evaluated through the following index: + color change detected, − no color change detected and + + color change detected throughout the entire medium.
Indole-3-acetic acid (IAA) production was determined by high performance liquid chromatography (HPLC; LC20 system, Shimadzu Corporation, Kyoto, Japan). Twenty ml of potato dextrose broth (PDB) medium (Becton, Dickinson and Company, MD, USA) was prepared in a 50 mL Erlenmeyer flask. RW309 was grown in PDB medium in triplicate for 7 days at 25 °C with or without 1 mM (final concentration) of tryptophan. After 7 days, the medium was collected and centrifuged for 10 min at 10,000×g. After that, 500 µL of PDB medium with or without tryptophan was transferred to a 1.5 mL tube and mixed with 500 µL methanol. For quantification of IAA, a standard curve was prepared with concentrations of 0.1, 1.0 and 10.0 mg L−1 of IAA. HPLC was conducted under the following conditions: flow rate of 1.0 mL min−1, Intersil ODS-SP column (4.6 × 250 mm), oven temperature of 40 °C, mobile phase of 70% of 50 mM potassium phosphate buffer (pH 7.0) and 30% of methanol, injection volume of 10 µL and detection by ultraviolet–visible detector at 280 nm.
The Carboxymethyl cellulose CMC (Nacalai Tesque, Inc, Kyoto Japan) plate assay was carried out to detect endo-glucanase activity (Colonia and Junior 2014). In briefly, 15 g L−1 of agar, 1% Carboxymethyl cellulose (CMC) mixture, 7.0 g L−1 of KH2PO4, 2.0 g L−1 of K2HPO4, 0.1 g L−1 of MgSO4 7H2O, 1.0 g L−1 of (NH4)2SO4, 0.6 g L−1 of yeast extract, 10 g L−1 of microcrystalline cellulose, and plates were poured by culture media. RW309 was grown on culture media in triplicate and incubated at 30 ºC for 4 days, and growth developed on culture plates. The culture was then flooded with 10 mL 0.1% congo red dye (Nacalai Tesque, Inc, Kyoto Japan) and stained for 30 min. After staining, the culture surface was washed with 5 mL 0.5 M NaCl for 10 minutes. Changes were evaluated by the appearance of a pale yellow halo zone surrounding the RW309 colony.
Colloidal chitin was prepared from commercial chitin crab shells powder by (Nacalai Tesque, Inc, Kyoto Japan). 10 g of chitin powder was added into 90 mL HCl and kept in a refrigerator (4 ºC) for 24 h. Then, 100 mL of distilled water was added and gelatinous white material formed was separated by filtration through glass filter paper. The obtained material was washed with distilled water via centrifugation until the filtrate had a pH 6.5. The final product of colloidal chitin obtained had a soft consistency, with 90% moisture (Rojas-Avelizapa et al. 1999). Later chitinase detection medium was prepared with the addition of colloidal chitin. The medium contains 0.3 g L−1 of MgSO4.7H2O, 3.0 g L−1 of (NH4)2SO4, 2.0 g L−1 of KH2PO4, 1.0 g L−1 of citric acid monohydrate, 15 g L−1 of agar, and 4.5 g L−1 of colloidal chitin; pH was adjusted at 4.7 and then autoclaved. The medium was poured in petri plates and allowed to solidify. RW309 plugs were inoculated in triplicate into the medium and incubated at 25 ºC for 4 days to be tested for chitinase activity. Results were compiled through the following index: + detectable clear zone, − non-detectable clear zone.
A pure culture of RW309 in triplicate was prepared to measure the phosphatase and β-glycosidase enzyme activities. The rest of the procedure was the same as the soil enzyme activities assay.
Incubation experiment
300 g of air-dried soil sample was transferred to Neubauer pots (100 cm2; Fujiwara Seisakusho, Ltd. Tokyo, Japan). Four treatments were prepared as follows: poultry compost (PC), poultry compost + RW309 (PCT), cattle compost (CC), cattle compost + RW309 (CCT). Three replicate pots were prepared for each treatment. Organic materials and mineral fertilizer (ammonium sulfate) as one of control were added at the amount of 200 mg N kg−1, which referred to the percentage of different total N applied from each source (the percentage of N for each different amendment is shown in Table 1). 1 ml of spore suspension (3.1 × 109 cfu ml−1) was inoculated into soil treatments with RW309 (PCT and CCT). The experiment was conducted for 12 weeks at 25 °C in the dark, and pots were covered with aluminum foil. Soil moisture content was maintained at 60% of water holding capacity of soil by monitoring twice a week and adjusted by the addition of deionized water. Sampling was performed at 0, 7, 14, 21, 28, 66 and 99 days after addition of amendments and parameters were recorded at each sampling date, including inorganic N, enzyme activities, soil moisture and microbial abundance (Trichoderma spp. and other fungi were separately counted based on their morphological feature).
Inorganic N in soil
To measure inorganic N, 5 g of soil was extracted using 50 mL of 1 M KCl with shaking for 30 min. The extract was filtered (Advantech 110 mm filter paper, Toyo Roshi Kaisha, Ltd., Japan) and then the extract was stored at − 80 °C until further analysis. The components of inorganic N (NH4+-N and NO3−-N) were measured using the indophenol blue and alkali reduction diazo dye methods (Soil Environmental Analytical Method, 1997), respectively.
N mineralization in treatments (PC, PCT, CC, CCT) during the incubation period was calculated using the following formula:
*Soil indicates the treatment with no organic materials amendment, and total N is the total N from organic materials calculated from the analyzed value.
Soil enzyme activities
Soil phosphatase and glycosidase activities were measured using 0.5 g of soil sample from each treatment. Soil samples were taken and placed into 15 mL conical tubes, then 1.5 mL of buffer solution (0.2 M sodium hydrogen phosphate and 0.1 M of citric acid, pH 4.9, for glycosidase and 0.5 M of malic acid and 0.5 M of tris (hydroxymethyl) aminomethane, pH 6.5, for phosphatase), 0.1 mL of toluene, 0.9 mL of distilled water and 0.6 mL of 50 mM p-nitrophenyl-β-D-glucopyranoside monohydrate or 50 mM p-nitrophenyl phosphate disodium solution were then added into glycosidase and phosphatase tubes, respectively. Tubes were shaken vigorously for 30 s and then incubated at 35 °C for 1 h. After incubation, 8 mL of ethanol was added and tubes were shaken again for 30 s. After shaking, the soil suspensions were filtered through filter paper (Advantech 110 mm). One mL of filtrate was taken and mixed with 2 mL of 2 M Tris buffer and absorbance was determined by spectrophotometer at 400 nm. A standard curve was obtained using 0, 20, 50 and 200 µM of p-nitrophenol solutions. Control tubes were also prepared to compensate for any background absorbance not related to p-nitrophenol released by glycosidase or phosphatase activity (Kataoka 2017).
Colonization of Trichoderma sp. strain RW309 in soil
For the fungi isolation, 0.5 g soil samples were mixed with 5 mL of sterile distilled water via vortexing, and then a series of tenfold dilutions (10−2, 10−3, and 10−4) were made. 50 µL of the resulting soil suspensions were then plated onto Martin agar plates (Martin 1950) and the plates were incubated at 25 °C for 5 days.
Pot experiment for plant growing
Another pot experiment was carried out using the same organic materials as used in the incubation study, and oil cake was used as one of controls. The soil was prepared by the addition of phosphate (150 mg kg−1 of soil) as mineral fertilizer, and then the organic materials were mixed into the soil. All pots were covered with aluminum foil and incubated for 2 weeks at 25 °C to stabilize microbial activity. After 2 weeks pre-incubation, B. rapa was grown in the pots for 5 weeks in a growth chamber at 25 °C, with three replicates (under the same conditions as described above). B. rapa was harvested after 5 weeks and the above- and below-ground biomass was determined.
Fungal diversity and community composition
Soil samples were taken from the rhizosphere soil after plants were harvested from the pot experiment and stored at − 80 °C until further analysis. DNA was extracted from 0.5 g soil using the Fast DNA Spin Kit for Soil DNA Extraction (Bio 101, Vista, CA, USA) according to the manufacturer’s instructions. The concentration and quality of extracted DNA samples were measured using a BioSpec-nano spectrophotometer for life science (Shimadzu Corporation). The extracted DNA was used as template for polymerase chain (PCR) reaction of the internal transcribed spacer (ITS) region using the primers ITS1/ITS2 to amplify the ITS region for fungal community through additional barcode sequences. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). The qualification and quantification of PCR product was measured by mixing same volume of 1 × loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2 percent agarose gel for detection. Samples with bright main strip between 400 and 450 bp were chosen for further experiments. Later, the PCR products were mixed in equidensity ratios. Then, mixture PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany). The libraries—250 bp paired-end reads generated with NEBNext® UltraTM DNA Library Prep Kit for Illumina and library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Paired-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired-end reads were merged using FLASH (V1.2.7,http://ccb.jhu.edu/software/FLASH/) (Magoč and Salzberg 2011). Quality filtering on the raw tags were performed under specific filtering conditions to obtain the high-quality clean tags (Bokulich 2013) according to the QIIME(V1.7.0, http://qiime.org/index.html) quality controlled process (Caporaso 2010). The tags were compared with the reference database (Gold database, http://drive5.com/uchime/uchime_download.html) using UCHIME algorithm (UCHIME Algorithm, http://www.drive5.com/usearch/manual/uchime_algo.html) (Edgar et al. 2011) to detect chimera sequences (https://drive5.com/usearch/manual/chimeras.html). Next the chimera sequences were removed (Haas 2011) and finally, effective tags were obtained. Sequences analysis were performed by Uparse software (Uparse v7.0.1001 http://drive5.com/uparse/) through using final effective tags (Edgar 2013). Sequences with ≥ 97% similarity were assigned to the same OTUs. Representative sequence for each OTU was screened for further annotation. Sequences analysis were performed by blast with Qiime (Version 1.7.0, http://qiime.org/scripts/assign_taxonomy.html) and and Unit database (https://unite.ut.ee/) for species annotation at each taxonomic rank (Altschul et al. 1990; Kõljalg et al. 2013). The phylogenetic relationship of different OTUs, and the difference of the dominant species in different treatment, were conducted using the MUSCLE software (Version 3.8.31, http://www.drive5.com/muscle/) for multiple sequences comparison (Edgar 2004). The information of OTUs abundance was normalized using standard sequence number corresponding to the sample with the smallest sequences. Subsequent analysis was achieved based on the normalized output data.
The reads were submitted to DDBJ Sequence Read Archive (https://www.ddbj.nig.ac.jp/dra/index-e.html) under Bioproject and are available under accession number DRA010896.
Statistical data analysis
Student t test and Dunnett’s (http://www.gen-info.osaka-u.ac.jp/MEPHAS/mokuji1-e.html) test were conducted for single and multiple comparisons of pot experiments (Mahmood et al. 2019). Mothur software was used to analyze the alpha diversity index (Uroz, 2016) and the coverage index was used to signify the sequence depth. Shannon-Weiner index (http://www.mothur.org/wiki/Shannon) was used to calculate the community diversity.
Results
Plant growth promoting ability of Trichoderma sp. strain RW309
B. rapa and lettuce grown with RW309 showed significantly increased dry shoot and root biomass when compared with those without RW309 (Fig. 1). The shoot and root biomass of B. rapa was 17.6% and 73.8% higher, respectively, when compared with those of the control, whereas the shoot and root biomass of lettuce was 28.5% and 64.6% higher, respectively, when compared with the control.
Strain RW309 traits
Plant growth promoting (PGP) traits were tested and revealed that RW309 did not show phosphate solubilization or siderophore production (Table 2). However, the IAA concentration was significantly higher in RW309 spent broth with tryptophan than that in the control with tryptophan. The concentrations of IAA were 12.3 and 2.19 mg L−1 in RW309 spent broth with and without tryptophan, respectively. Furthermore, RW309 was characterized to measure the enzyme production, and RW309 showed a pale yellow halo zone surrounding the RW309 colony in terms of endo-glucanase activity, but not for chitinase. Moreover, phosphatase and β-glycosidase activity was 7.88 and 33.2 Units h−1, respectively (Table 2).
Incubation assay
N mineralization
N mineralization was measured throughout the 12 weeks of incubation. Inorganic N in PC and PCT was released at 14% and 4%, respectively, at the start of the incubation, which subsequently increased to 49% and > 100%, respectively, at day 99 of incubation. Inorganic N release in PCT was significantly higher than that in PC at day 99 of incubation (Fig. 2). Inorganic N in CC and CCT was 42% and 31%, respectively, at the start of the incubation, which subsequently increased to 51% and 96%, respectively, at day 99 of incubation. Inorganic N release in CCT was significantly higher than that in CC at days 66 and 99 of incubation (Fig. 2).
Soil enzyme activities
The soil enzyme activities of phosphatase and glycosidase were measured during the incubation experiment. Phosphatase activity was 11.8 and 8.7 ng · g−1 of soil in PC and PCT, respectively, at the start of the incubation, and increased to 56.5 and 68 ng · g−1 of soil, respectively, at day 66 of incubation (Fig. 3a). Phosphatase activity was significantly higher in PCT than that in PC at days 66 and 99 of incubation. Phosphatase activity was 10.4 and 9.35 ng · g−1 of soil in CC and CCT, respectively, at the start of the incubation, and increased to 63.5 and 70.2 ng · g−1 of soil, respectively, at day 66 of incubation. Phosphatase activity was significantly higher in CCT than that of CC and the control at day 66 of incubation.
In contrast, glycosidase activity was low at the start of incubation, but the activity increased during the incubation period (Fig. 3b). Glycosidase activity was 12.4 and 15.2 ng · g−1 of soil in PC and PCT, respectively, at the start of the incubation, and increased to 33.1 and 35.1 ng · g−1 of soil, respectively, at day 99 of incubation. Glycosidase activity of PCT was slightly higher than that of PC and significantly higher than that of the control. Glycosidase activity was 15.4 and 15.6 ng · g−1 of soil in CC and CCT, respectively, at the start of the incubation, and increased to 28.5 and 30.6 ng · g−1 of soil, respectively, at day 99 of incubation. Glycosidase activity of CCT was slightly higher than that of CC and significantly higher than that of the control.
Colonization of RW309 in soil
The abundance of total fungi and Trichoderma spp. was counted separately based on their morphological characteristics. There was no change in total fungal cfu in the N (mineral fertilizer) and S (only soil without any organic matter) treatments. The fungal abundance increased gradually after day 21 in both PC and CC treatments. Fungal abundance was higher in PCT and CCT than in PC and CC, respectively. Furthermore, a difference in the growth rate of Trichoderma spp. was confirmed between CCT and PCT. The growth rate of Trichoderma spp. was highest on day 14 in CCT, and on day 28 in PCT (Fig. 4). The Trichoderma spp. abundance was higher in CCT than in PCT throughout the incubation period.
Pot experiment
All treatments enhanced plant growth (dry weight of shoot and root biomass) when compared with the control (Fig. 5a). The dry weight of PCT was significantly higher than that of PC (t-test, P < 0.05), but there was no significant difference in dry weight observed between CCT and CC. In contrast, there was a significant different in the fresh weight between CC and CCT whereas there was no significant difference between PC and PCT (Fig. 5b).
Fungal diversity and community composition
A total of 686,900 sequences and the maximum numbers of species per sample were obtained for all treatments. Fungal community composition showed substantial differences among treatments. The relative abundance of fungal families is shown in Fig. 6a. The Shannon–Wiener diversity index (H’) reflected the effect of RW309 on fungal diversity (Table 3). However, differences were observed between PC and CC. The diversity in CC was higher (H’ = 4.637) than that in CCT (H’ = 3.109), while the diversity in PC (H’ = 4.482) was lower than that in PCT (H’ = 4.528). Furthermore, Chao1 index also reflected the effect of RW309 on fungal diversity and differences were observed between PC and CC.
Principal coordinates analysis (PCoA) of the unweighted Unifrac a distances was used to determine the association between the fungal community and treatments for β diversity analysis (Fig. 6a). The first two principal coordinates signified 23.09% (PC1) and 22.67% (PC2) of the soil fungal community variation. According to PCoA, S, N and OC are clearly separated from CC, CCT and PC, PCT plots. Also there are differences between CC and CCT, and between PC and PCT although the each treatment were used same substrate. This indication reflected fungal community structure had changed substantially with RW309 when inoculated with different substrates. Furthermore, a noticeable difference was observed between CCT and PCT plots, indicating that manure types also had important effects on the soil fungal community composition.
The dominant fungal families throughout the treatments were Hypocreaceae, Chaetomiaceae, Nectriaceae, Incertae sedis Hypocreales, Pezizaceae and Lasiosphaeriaceae. The relative abundance of family Hypocreaceae was highest in CCT and PCT when compared with the other treatments. However, family Chaetomiaceae was dominant only in CC and PC (Fig. 6b).
Discussion
This study investigated the possibility of plant growth promotion with Trichoderma sp. strain RW309 inoculation. Plants grown in CC had greater biomass than those grown in PC in a pot experiment.
Trichoderma spp. enhanced nutrient availability from the rhizosphere and plant nutrient uptake and assimilation (Sani et al. 2020). Nutrients in soil can occur in slightly insoluble or soluble forms, which can restrict the circulation of nutrients in soil. Trichoderma spp. have the ability to change the pH value of soil as well as the utilization and recycling of organic nutrients into inorganic nutrients that are plant available (El-Katatny 2010; Hermosa et al. 2012; Li et al. 2015). Accessibility of nutrients in the rhizosphere could also be directly or indirectly affected by Trichoderma spp., which are able to solubilize nutrients like Fe and P (Li et al. 2015). Trichoderma spp. could transform organic N into inorganic N forms and discharge low amounts of NO2 (Maeda 2015). N mineralization differed between the composts (poultry and cattle compost) used in this study. The type and age of animal manure compost can have different effects on N mineralization (Chadwick et al. 2000), because organic composts stimulate microbial growth and amplify competition for inorganic nutrients (Cordovil et al. 2005). Organic composts inoculated with RW309 showed positive effects on N mineralization and soil enzyme activities. N mineralization was accelerated in PCT and CCT when compared with PC and CC, respectively (Fig. 2). Thus, RW309 inoculation enhanced the degradation of organic materials, resulting in the release of plant available nutrients. There were no significant differences in glycosidase activity between organic materials with and without RW309, but at the end of incubation, glycosidase activity in organic materials with RW309 was slightly higher than in those without RW309 (Fig. 3b). In contrast, endo-glucanase was produced by RW309. In addition, higher phosphatase activity was observed in organic materials with RW309 than in those without RW309; similar results were also observed in pot experiments where phosphatase activity was higher in organic materials with RW309 than in those without RW309 (data not shown). Moreover, RW309 produced phosphatase and β-glycosidase even in pure culture (Table 2). Thus, it was suggested this higher value of soil enzymes derive from RW309. Thus, RW309 inoculation resulted in increased soil enzyme activities and enhanced nutrient accessibility for plant growth and soil improvement (Mbarki et al. 2017). Generally, Trichoderma spp. have the ability to secrete cell wall degrading enzymes such as cellulases, glucanases, chitinases, proteinases and xylanases, which can help with the degradation of microbial cells in the rhizosphere and lead to increased absorption of essential plant nutrients (Assigbetsé et al. 2012; Halifu et al. 2019). Therefore, our results indicated that the combination of organic materials with RW309 was optimal for enhancing enzyme activities and contributing to the degradation of organic compounds.
Trichoderma spp. grow quickly in soil, and our results suggested that CC was more suitable for culture of RW309 growth than PC. For sustainable agriculture, fungal diversity is important for maintaining soil functions, which reduced fungal diversity is often observed with excessive mineral fertilizer use (Keszthelyi et al. 2008; Bärlocher and Boddy 2016; Cai 2019). Ye et al. (2020) found that organic fertilizers inoculated with Trichoderma spp. showed higher abundance of fungi, bacteria and actinomycetes. Ideally, RW309 inoculation would activate soil microbes and increase their diversity, however, our results showed that RW309-dominated CCT, PCT had reduced alpha and beta diversity (Table 3), which was similar to the results of previous studies (Raaijmakers et al. 2009; Pang 2017; Zhang 2018; Zhang et al. 2019). Trichoderma spp. have the advantages of vigorous vitality and fast growth; thus, their strategy sometimes shows to inhibit the growth space faster and to absorb the necessary nutrients. In this study, however, CCT and PCT inoculated with RW309 resulted in reduced abundance of genus Fusarium (Fig. S2), which can be the causal agent of some soil borne diseases (Wang et al. (2015); Larkin et al. (1996); Roberts et al. (2000). On the other hand, our results suggested that RW309 produced IAA during its growth. Normally, IAA production increases with fungal growth (França et al. 2017; Kumar et al. 2017). In this study, RW309 had high abundance in CCT at day 14 of incubation, and then decreased again (Fig. 4). In culture media, RW309 grew rapidly and produced IAA at the concentration of 12.3 mg L−1 in spent broth with tryptophan after 1 week of incubation. In addition to the growth of RW309 in soil, the effect of RW309 on plant growth promotion was revealed by the inoculation of RW309 alone and also in combination with organic materials. However, plant biomass results showed that there were opposite results for dry and fresh weight of biomass between CC/CCT and PC/PCT. For fresh weight, CCT had significantly higher shoot biomass than CC. It means that IAA produced by RW309 might contribute resulting in enhanced plant growth during the early stage in CCT. The plant hormone auxin is well known to stimulate cell elongation via increasing cell wall extensibility (Majda and Robert 2018), which serves as a distribution route for water uptake (Petrášek and Friml 2009). Therefore, this might explain why the fresh weight of CCT was higher than that of CC.
Conclusion
This study showed that inoculation with Trichoderma sp. strain RW309 enhanced plant growth of B. rapa and lettuce. Both incubation and pot experiments showed that inoculation of RW309 with organic materials increased N mineralization and soil enzyme activities. Addition of organic materials with RW309 changed the fungal community in soil. The results suggested that this combination of organic materials with RW309 has great potential for plant growth enhancement during the early and late stages of plant growth through the release of secondary metabolites and increased nutrient availability.
Availability of data and material
All data generated or analyzed during this study are included in this published article. In addition, sequencing data were deposited to DNA Data Bank of Japan (DDBJ).
Code availability
All software are available.
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Acknowledgements
This study was supported by Nippon Life Insurance Foundation. We thank to Mr. Yohei Mukouyama who provided poultry compost. We also thank Catherine Dandie, PhD, from Edanz Group (https://en-author-services.edanzgroup.com/ac) for editing a draft of this manuscript.
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This study was supported by Nippon Life Insurance Foundation.
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Conceptualization, RK; methodology, RK; WA; writing—original draft preparation, RK; WA, writing—review and editing, RK; visualization, RK, supervision, RK; project administration, RK; funding acquisition, RK. All authors have read and agreed to the published version of the manuscript.
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Asghar, W., Kataoka, R. Effect of co-application of Trichoderma spp. with organic composts on plant growth enhancement, soil enzymes and fungal community in soil. Arch Microbiol 203, 4281–4291 (2021). https://doi.org/10.1007/s00203-021-02413-4
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DOI: https://doi.org/10.1007/s00203-021-02413-4