|Other Abstract||Trichoderma spp. are widespread in nature and have important application in agriculture. They can not only control phytopathogens but also colonize on the root surface of plants to promote their growth. With the prevalence of green agriculture, environment-friendly microbial agents are urgently needed. Marine-derived Trichoderma refers to the Trichoderma spp. obtained from marine environments. Due to the unique habitats, the research on marine-derived Trichoderma species and its secondary metabolites may have important significance.
Trichoderma asperelloides RR-dl-6-11 was isolated from the inner tissue of the marine alga Rhodomela confervoides. It was subjected to batch fermentation, and its total secondary metabolites were extracted. The monomeric compounds were separated by silica gel chromatography, reversed-phase chromatographic column, Sephadex LH-20 gel chromatography, preparative thin layer chromatography and HPLC. Their structures were further determined by NMR, IR, MS, ECD and X-ray. Effects on T. asperelloides RR-dl-6-11 resulted in the isolation and identification of 44 monomer compounds, with eighteen (1–18) including one rare proharziane diterpene (1), three harziane diterpenes (2–4), ten bisabolane sesquiterpenens (5–14), one cadinene sesquiterpenen (15) and three cyclonerane sesquiterpenens (16–18). Compound 4 features an opened ring A that has not been reported previously. Compound 16 represents the first occurrence of cyclopentenyl-bearing cycloneranes, and 18 seems a cyclopentyl-degrading cyclonerane derivative.
The crude extract and some monomeric compounds of T. asperelloides RR-dl-6-11 were evaluated for antimicroalgal and antimicrobial activities. The results showed that crude extract and sesquiterpenoids (5–18) exhibited significant inhibitory activity against Amphidinium carterae, Chattonella marina, Heterosigma akashiwo and Prorocentrum donghaiense, with IC50 values ranging from 0.54 to 13 μg/mL. Sesquiterpenoids were more active to four phytoplankton species than harziane diterpenes. Mechanistic studies demonstrated that the crude extract and compounds 5, 15, 20 and 23 changed the contents of superoxide dismutase, peroxidase, soluble protein, malondialdehyde, chlorophyll a and carotenoid in microalgae cells. They could disturb the oxidation-antioxidation balance in microalgae cells, cause lipid peroxidation of cell membrane, break down photosynthesis system, affect the cellular homeostasis, and eventually lead to cell death. The crude extract of T. asperelloides RR-dl-6-11 showed excellent inhibitory activity against Colletotrichum cingulate and Fusarium oxysporum f. sp. cubense with MIC values of 6.25 and 12.5 μg/mL, respectively. On the other hand, the inhibition of the marine bacteria Vibrio anguillarum, V. harveyi, V. parahemolyticus, V. splendidus, and Pseudoalteromonas citrea was also assayed, only the crude extract and some monomeric compounds had certain antibacterial activity.
Plate confrontation experiments of T. asperelloides RR-dl-6-11 with other algicolous fungi and plant pathogenic fungi indicated that T. asperelloides RR-dl-6-11 could effectively inhibit the tested fungi by growth competition, hyperparasitism and antibiosis. Enzyme activity experiments showed that T. asperelloides could secrete cellulase and chitinase extracellularly to break down the cellulose and chitin in Fusarium cell wall, and achieve the purpose of inhibiting the growth of Fusarium. In view of the significant antifungal activity of T. asperelloides RR-dl-6-11, the T. asperelloides was developed into T. asperelloides agent using by-products of agricultural products processing (chaff: bran = 2: 1) for agriculture in salt-alkaline land. The results of germination experiments on glass plates, pot culture experiments in phytotron and field experiments on wheat showed that the T. asperelloides agent had significant growth-promoting effects, resulting in remarkable increases in total root length, root surface area, root tip number, root projected area, mean root volume and stem length of wheat seedlings. Microbial high-throughput sequencing analysis was carried out on field soils treated with T. asperelloides agent. The results of the analysis revealed that T. asperelloides agent increased the microbial richness in the field soil, and the effects of T. asperelloides agent on bacterial community was greater than that on fungal community.
For the first time, the secondary metabolites of T. asperelloides RR-dl-6-11 have been investigated. The structural types cover diterpenes, sesquiterpenes, polyketides and steroids. We also evaluated their activities against red tide microalgae, agricultural plant pathogenic fungi and aquatic pathogenic bacteria, and explored the mechanisms of anti-microalgal action. These enriched the structural and active diversity of the secondary metabolites from marine-derived Trichoderma. Combined with further studies on the antifungal activity of T. asperelloides RR-dl-6-11, RR-dl-6-11 was developed as an agricultural agent and applied in salt-alkaline land. The agent has the dual role of growth-promoting effect on wheat and regulation microflora of rhizosphere, which provides an important reference for the application of marine-derived Trichoderma in saline and alkaline land.|