Among humic acids, fulvic acid is considered as a very promising biofertilizer. Fulvic acid can accelerate the growth rate of plants by increasing their metabolic and developmental activities (such as their photosynthetic efficiency and root growth rate) and enhancing their stress resistance (such as drought resistance). In addition, researchers have found that the quinone and phenolic structures in fulvic acid exert direct toxic effects on plant pathogenic fungi. It has already been proved that fulvic acid can reduce the incidence of partial pathogenic fungi, which usually belong to Ascomyces and Basidiomycetes, on plants (including the fungi belonging to Fusarium, Alternaria, Sphaerotheca and so on).
Rice (Oryza sativa L.) is considered one of the most important crops across the world, and is used as an energy source for over 50 % of the global population.The global population is expected to exceed 10 billion by 2050, which would require food output to increase by at least 70 %. Rice crops are, however, vulnerable to various biological stresses, which seriously threatening the world’s food security. Disease is the most significant factor affecting the yield and quality of rice, and the common diseases of rice crops include sheath blight, rice blast, rice seedling blight, bacterial blight of rice and so on.
Among them, rice sheath blight ranks first among the three major diseases, and is caused by the pathogen Rhizoctonia solani. At present, the prevention and control of rice sheath blight relies mainly on chemical fungicides. However, chemical fungicides are prone to ecological toxicity and soil microbial destruction, and may also lead to the emergence of drug-resistant pathogens. Therefore, biological control agents have emerged as an effective alternative to chemical fungicides.
As one of the most widely used species for biological control in the filed, Trichoderma asperellum has the characteristics of strong reproductive ability, superior survival ability and environmental friendliness. The use of certain biological extracts results in enhanced the efficacy of biological fungicides. Therefore, it is necessary to find a way to combine the biological extracts with biocontrol fungi together to enhance the ability of the biocontrol fungi for pathogenic fungi inhibition.
Effects of fulvic acid on the growth and relative enzyme activities of the two fungi
The effect of fulvic acid addition to a co-culture system for the antagonistic effect of Trichoderma asperellum YL127-02 on R. solani YL127-01 was investigated. The results showed that the inhibition zones of R. solani YL 127-01 could be observed on both of the control group and test groups. Interestingly, with the addition of fulvic acid, the antagonistic effect of Trichoderma asperellum YL127-02 against R. solani YL127-01 was much greater than that of in the control group. After four days of incubation, the inhibition rate of Trichoderma asperellum YL127-02 against R. solani YL127-01 was 78.28 %, whereas only a 57.93 % of inhibition rate was noted in the control group (Fig. 1A, B).
The strong antagonistic effect of Trichoderma asperellum YL127-02 against R. solani YL127-01 might be attributed to the different effects of fulvic acid on the biocontrol fungus and the pathogenic fungus of their growth and development, respectively. Thus, the coculture system was separated and fulvic acid was added to each pure culture to investigate the promotion or inhibition effects of fulvic acid on the different fungi.
As shown in Fig. 1C, the mycelial colonies of R. solani YL127-01 treated with fulvic acid were smaller than those in the control group, and the maximum growth inhibition rate of fulvic acid on R. solani YL127-01 growth reached to 18.68 %. Interestingly, the radius of Trichoderma asperellum YL 127-02 in the test group was above 50 % larger than that of in the control group (Fig. 1D). The results clearly indicated that the fulvic acid could enhance the antagonistic effects of Trichoderma asperellum YL127-02 against R. solani YL127-01 by promoting the growth of the biocontrol fungus and inhibiting the growth of the pathogenic fungus, simultaneously.
The oxidation-antioxidant statuses inside the cells of the two fungi were detected. NOX, a key enzyme in ROS production, showed maximum activity (229.58 U/mg pro-1) after a 96 h incubation of R. solani YL 1207-01, which was 22.82 % higher than that of in the control group (Fig. 2A). Meanwhile, the H2O2 and O2·– levels inside the pathogenic fungal cells in the test group were 62.81 % and 37.03 % higher than those of in the control group, respectively (Fig. 2B). The above results indicated that R. solani YL 1207-01 suffered from high oxidative stress when fulvic acid was added to the culture system.
In addition, the pathogenic fungus could not cope well with the increased oxidative stress. The activities of a series of enzymes related to oxidant elimination (GST, CAT and SOD) were found to decrease by 30.57 % to 59.03 %, respectively, in the the test group after a 96 h incubation (Fig. 2C), indicating that the ROS elimination system of the pathogenic fungus had already collapsed. Thus, the oxidation-antioxidant balance of fungal cells was finally broken, and some of the crucial metabolic pathway in the cells of the pathogenic bacteria were disrupted. For example, the activities of HK and G6P, which catalyze glucose phosphorylation in glycolysis and gluconeogenesis, declined over time in R. solani YL127-01 cells.
After 96 h, the test group showed only 90 % and 70 % relative HK and G6P activity, respectively, compared to the control group, which suggests fulvic acid had disrupted the fungus’s carbohydrate metabolism (Fig. 2D). Moreover, the MDA content in the supernatant of R. solani YL127-01 culturing system in the test group was much higher than that in the control group, indicating that some fungal cells had already been disrupted under the high level of oxidative stress.
Surprisingly, the activities of the antioxidant enzymes of Trichoderma asperellum YL127-02 were completely opposite compared with those of R. solani YL127-01 when fulvic acid was added to the culturing system. Compared with the control group, the levels of three enzymes, SOD, POD and PPO, increased by 55.79 %, 51.04 % and 43.76 %, respectively, in the test group, resulting in the lower level of ROS content in the test group (Fig. 3A and 3B). The results indicated that Trichoderma asperellum YL127-02 could relieve the oxidative stress by increasing the activities of the antioxidant enzymes to maintain its oxidation-antioxidant balance inside its cells.
Moreover, fluvic acid stimulated Trichoderma asperellum to secrete higher levels of cellulase, chitinase, and β-1,3 glucanase. The final activities of the three enzymes increased of 40.13 %, 78.46 % and 79.39 %, respectively, in the test group compared with those of in the control group after a 96h incubation (Fig. 3C). The higher activities of the three enzymes could give the biocontrol fungus higher efficiency on destroying the cell wall of the pathogenic fungus.
Transcriptome analysis
The changes of gene transcription in R. solani YL127-01 and Trichoderma asperellum YL127-02 with or without fulvic acid addition were analyzed and the possible metabolic pathways in the two fungal strains influenced by fulvic acid were detected through the transcriptome analysis. A total of 12 samples were sequenced and the initial transcriptome data were acquired.
After filtering, 138.47 M and 138.8 M clean reads were obtained from the R. solani YL127-01 samples with or without fulvic acid addition, while 140.86 M and 141.74 M clean reads were obtained from the test and control groups of Trichoderma asperellum YL127-02. The quality of data assessment showed that Q20 and Q30 ratios of all the samples were greater than 97 % and 96 %, respectively, and the GC counts of each sample ranged from 52.21 % to 54.81 %.
Based on the DESeq standardization method, with q-value < 0.05 and |log2|(fold change)| > 1 as thresholds, differential gene expression was analyzed in different treatment groups. In comparison to the control group, a total of 7539 and 5548 DEGs were identified in R. solani YL127-01 and Trichoderma asperellum YL127-02 treatment groups, of which 4496 and 2486 genes were upregulated while 3043 and 3062 genes were downregulated, respectively (Fig. 4).
The GO analysis showed that the DEGs of R. solani were divided into 533 subcategories, including molecular function items, biological process items, cell components items and so on. Among them, the down-regulated genes were mainly involved in “ribosomal structure composition”, “NAD binding”, “redox process”, and “carbohydrate metabolism process”. Meanwhile, DEGs of Trichoderma asperellum were annotated into 374 subcategories. The most DEGs were related to molecular functions and biological processes. Specifically, the up-regulated genes in the treatment group were mainly involved in “oxidoreductase activity”, “peroxidase activity”, and “glutamine tRNA (Gln) aminotransferase complex”.
KEGG annotation indicated that 2267 DEGs of R. solani YL127-01 were annotated and classified into 37 groups comprising 205 functional categories based on the secondary pathway hierarchy. Among them, 851 identified genes related to crucial metabolic processes mainly belonged to 37 categories were found to have lower expression rates in the test group than in the control group (Fig. 5A).
The first type of gene with a relatively low expression rate in the R. solani test group was related to the energy and carbohydrate metabolism. The expression rates of the genes encoding the enzymes that are responsible for catalyzing of glucose oxidation (e.g. 6-phosphofructokinase, hexokinase and fructose-bisphosphate aldolase), were obviously decreased in the test group. The lower expression rate of the gene encoding riboflavin synthase was also found in the test group, indicating that the fungal cells might have low FAD and FMN production. A series of genes belonging to the Nuo gene family, which are responsible for theconstruction of NADH dehydrogenase complex, had low expression rates in the test group.
In addition, the expressions of the genes which are related to cytochrome c oxidase assembly were decreased in the test group, resulting in the low efficiency of electron transfer to the mitochondrial membrane of the cells. The results mentioned above clearly indicated that fulvic acid had greatly interfered with the ability of pathogenic fungal cells to catabolize carbohydrates, resulting in fewer metabolites and reducing power could be produced and be used for anabolic pathways. Moreover, the respiratory chain was not well developed inside the cells of the test group, indicating that less energy could be generated through oxidative phosphorylation process. Fewer metabolites and a lower energy supply strongly inhibited the growth and development of the fungal cells.
The second large group of differentially expressed genes in the test group of R. solani are related to protein synthesis. Numerous genes encoding ribosomal proteins small subunit (RPS) and ribosomal proteins large subunit (RPL) were down-regulated in the fungal cells of the test group, which might have severe negative effects on the subsequent protein synthesis process. For example, the down-regulation of RPS3, RPS13 and RPS16 decreased the binding efficiency of EIF-2-GTP-initiating tRNA complex to ribosome small subunits, and decreased the binding efficiency of initiation complex to mRNA 5′ end, resulting in the low efficiency to start the initiation process of peptide chain synthesis.
The down-regulation of RpL12, RPS13, RPL3 and other ribosomal proteins interfered with the efficiency of the processes related to the entry of aminoacyl tRNA into the ribosome A site, the binding of codons and anticodons and the rate at which the ribosome moves along the mRNA, which greatly reduces the elongation rate of the peptide chain. Moreover, the low expression rates of RPS7 and RPS12 indicted that the fungal cells group might have difficulties on protein folding and transport. The results mentioned above showed that fulvic acid interfered with the whole process of protein translation of the pathogenic fungus.
The third group of genes with lower expression rate in the test group was related to ROS elimination. The genes encoding the enzymes of SOD and CAT were down-regulated by 50 % and 40 %, respectively, in the test group. Owing to the low expression of these enzymes, the oxidation-antioxidant balance within the fungal cells was disrupted gradually. Thus, ROS would be accumulated inside the cells, resulting in severe oxidative damage to cellular components.
The above results indicated that fulvic acid could inhibit the metabolic processes, the energy metabolism and biosynthesis of key cellular substances of the pathogenic fungus, disrupting the “oxidation-antioxidant balance” inside the fungal cells. Thus, it was not difficult to conclude that the growth of the pathogenic fungus would be severely inhibited by fulvic acid.
This conclusion could also be confirmed by the transcriptome results. It could be observed that the expression rate of the cell cycle regulatory protein CDC2 was decreased in the test group, indicating that the cells might be arrested in the G2 phase, which significantly prolonged the duration of the cells mitosis process. A low expression rate of CDC37 was also observed, which might inhibit the key regulatory kinase (CDC28) for major cell cycle transitions, leading to cell cycle arrest.
To our surprise, fulvic acid did not have obvious negative effects on the fungal cells of Trichoderma asperellum YL127-02. Genes related to crucial functions (such as protein synthesis and DNA replication) were expressed regularly. Moreover, some genes related to key metabolic pathways of Trichoderma asperellum were up-regulated in the test group, indicating that these cells might have a relatively higher metabolic activity..
As shown in Fig. 5B, the most important genes which were up-regulated in the test group are related to metabolism of organic carbon. The genes encoding glucose-6-phosphate isomerase, succinate dehydrogenase, and glucose-6-phosphate dehydrogenase were up-regulated by at least two times, indicating that the efficiency of the crucial pathways related to glucose catabolism (including glycolysis, tricarboxylic acid cycle and pentose phosphate pathway) was greatly improved.
Moreover, the expression levels of acetyl-CoA synthetase and pyruvate carboxylase in the test group were 2.1 and 2.2 times higher than those in the control group, indicating that the fungal cells in the test group had the strong ability to catalysis key intermediate metabolites production (such as oxaloacetic acid and acetyl-CoA) within a shot period, which could effectively connect the catabolic and anabolic pathways inside the fungal cells.
Besides SOD and CAT, it was also found that the fungal glutathione system functions as another antioxidant mechanism to cope with the oxidative stress. A series of genes belonging to GST gene family were up-regulated by 3 to more than 14 times in the test groups than those of in the control groups. At the same time, glutathione peroxidase activity was increased by 14.28 times in the test group. With the multiple antioxidant defense systems, Trichoderma asperellum could cope well with the increase of oxidative pressure and maintain their oxidation-antioxidant balance.
The above results indicate that fulvic acid promoted the metabolism of carbohydrates inside the cells, which supplied the fungal cells with abundant energy, reducing power and metabolites for cell development, and it might lead to the rapid growth of the mycelia in the test group. Evidence of the rapid growth of fungal cells could also be found in the transcriptome results. The up-regulation of CDC20 might accelerate the transition of cell mitosis from the metaphase to the anaphase. Moreover, the up-regulation of anaphase-promoting complex (APC) expression level could enable the fungal cells to enter the G1 phase quickly. The results indicated that fulvic acid can accelerate the cell cycle of Trichoderma asperellum, thereby increasing its growth rate.
DEG validation through qRT-PCR analysis
To verify the credibility and accuracy of DEGs identified from transcriptome sequencing, some of the related functional genes were selected for qRT-PCR evaluation, respectively. The selected genes of R. solani YL127-01 were involved in the pathways of carbohydrate and energy metabolism (pfkA, NDUFV2 and ribE, encoding 6-phosphofructokinase, NADH dehydrogenase and riboflavin synthase, respectively), protein synthesis (rpsM, encoding ribosome small subunit protein S13) and ROS elimination (encoding the enzymes of CAT, SOD and GST).
It could be observed that the genes of pfkA, NDUFV2 and ribE in the test group with fulvic acid addition were decreased by 60.83 %, 46.28 and 80.75 %, respectively, compared with the control group (Fig. 6A). Meanwhile, the expression of rpsM gene was down-regulated by up to 88.99 % in the test group compared with the control group. Moreover, all the test antioxidant genes of R. solani YL127-01 were restrained when the fungus was threatened by the fulvic acid.
The four selected genes of Trichoderma asperellum YL127-02, pgi, G6PD, PC and ACU5, encoding the key enzymes which were responsible for glucose catabolism and intermediate metabolite synthesis presented high expression levels in the test group (Fig. 6B). Moreover, the high level expression of the antioxidant enzymes were also confirmed in the test group.
The above results demonstrated that all the test genes displayed a consistent expression pattern with the transcriptome sequencing results, which confirmed that the RNA-seq data was reliable and accurate.
Conclusion
The addition of prepared fulvic acid significantly improves the antagonistic effects of biocontrol fungus against pathogenic fungus. Further studies confirmed that fulvic acid had totally opposite effects on the metabolism and growth of the rice pathogenic fungus, R. solani and the biocontrol fungus, Trichoderma asperellum.
On the one hand, fulvic acid enhanced the level of oxidative stress of R. solani, which leads to a break oxidation-antioxidant balance inside the cells. Under this condition, some crucial metabolic pathways, including the pathway relative to intermediate metabolite production, energy and reducing power generation, and protein synthesis, were disrupted seriously, resulting in low level of fungal growth and development, even led to cell death.
On the other hand, Trichoderma asperellum could afford the additional oxidative stress by activating its antioxidant enzyme system to eliminate ROS. Moreover, fulvic acid accelerates the efficiency of carbohydrate catabolism in Trichoderma asperellum, resulting in the enhancement of the supply of intermediate metabolites and energy during the anabolic process of the fungal cells.
We supply raw materials. You own formulations. If you would like to learn more about fulvic acid and Trichoderma asperellum, please feel free to contact DORA team.