Identification and mechanism of Trichoderma longibrachiatum metabolites in inhibiting the Alternaria tenuissima toxin production

Abstract: 【Objective】Alternaria spp. can cause a variety of apple leaf diseases, which occur in all major apple producing areas in the world. It can lead to brown disease spots in apple leaves and even result in early defoliation, which seriously affects the development of apple industry and causes huge economic losses. Apple leaf blight caused by Alternaria tenuissima was found for the first time in apple producing areas of Gansu province. This fungus can damage apple leaves and petioles, causing leaves to die and fall off. A. alternata mainly damages plants by producing Alternaria toxins. The Alternaria toxins that have been found can be divided into five categories, i. e, diphenyl-α-pyrones and their derivatives, perylenequinones and their derivatives, tetraamino acids and their derivatives, long-chain amino polyols of glycerol tricarboxylate compounds, and hybrid structures. All of them have obvious toxicity, which can cause serious harm to plants and endanger the food safety of agricultural products. At present, chemical control is still the main means to prevent and control the diseases caused by Alternaria fungi. However, due to the influence of Alternaria resistance and environmental pollution, it is of great significance to find a series of biocontrol agents with higher controlling effect on Alternaria. Biocontrol of Trichoderma is safer and greener than the traditional chemical control methods. Trichoderma metabolites are also good antifungal substances, with broad-spectrum and efficient antibacterial activity, inhibiting the growth and metabolism of pathogenic fungi. The growth and metabolism of A. tenuissima ABL2 strainwere inhibited by Trichoderma longibrachiatum SC5 metabolites. The content of non-host selective toxins in the metabolites of A. tenuissima ABL2 strain and the relative expression of genes related to the synthesis of A. tenuissima toxin were determined. The inhibition mechanism of T. longibrachiatum SC5 metabolites on A. tenuissima ABL2 strain production was clarified. The aim was to provide a reference and theoretical basis for the prevention and control of apple leaf blight caused by A. tenuissima.【Methods】T. longibrachiatum SC5 and A. tenuissima ABL2 strains were cultured on PDA medium for 7 days. The PDB liquid medium was made, and the SC5 strain was cultured on the medium for 15 days. The fermentation broth was filtered with filter paper and the broth was retained. The liquid was added with 3- fold- volume- ethyl acetate and oscillated for 1 hour. After standing extraction, the organic phase was evaporated in a rotary evaporator, and 1 mL of methanol was added to dissolve and evaporate. The SC5 metabolites were made into an orginal liquid with a concentration of 200.00 mg·mL^-1 , and added to the PDA medium to make a drug-containing medium with different concentrations of SC5 metabolites (0.01, 0.05, 0.10, 0.25, 0.50, 1.00 and 2.00 mg·mL^-1 ). The ABL2 strain was inoculated on the drug-containing medium and cultured for 24 hours in a light incubator. The diameter of ABL2 colonies was measured and the colony inhibition rate was calculated on the 2, 4, 6, 8 and 10 days by cross method. On the 6th day, 5 mycelial plugs were prepared with a sterile puncher and placed in a 10 mL of EP tube, with 3 replicates per concentration. After adding 8 mL of ethyl acetate, ultrasonic oscillation was performed for 1.5 hours, centrifuged and filtered. After the ethyl acetate phase was evaporated to dryness, 1 mL of methanol was added to dissolve it, and 5 mm circular sterile filter paper was placed in it for later use. Inoculate healthy leaves after disinfection and rinsing using stab inoculation method. A circular filter paper soaked in ABL2 metabolites was placed at each wound, and placed in an artificial climate box for moisturizing culture for 7 days. The lesion size was measured and the pathogenic activity of the metabolites was calculated. The optimal inhibitory concentration of SC5 metabolites on the growth of ABL2 strain was screened. The total RNA and the metabolites of ABL2 strain growing in the optimal concentration of drug-containing medium was extracted on 2, 4, 6, 8 and 10 days, and the quantitative standard curves of six non-host-selective toxins were made respectively. Six toxin-producing related gene primers were designed and verified for specificity. Liquid Chromatograph Mass Spectrometer and Real Time Quantitative PCR were used to determine the content of toxins in ABL2 metabolites and the relative expression of toxin-producing related genes.【Results】The crude extract of SC5 metabolism at a concentration of 0.5 mg·mL^-1 had a significant inhibitory effect on the growth and pathogenicity of ABL2, and the inhibition rates were 38.08% and 76.96% on the 6th day, respectively. 0.5 mg ·mL^-1 SC5 metabolic crude extract had a significant inhibitory effect on the non-host selective toxins TEN, ALT and TeA produced by ABL2 strain. The inhibitory effect was the most significant after 2 days treatment, and its content was reduced by 69.39%, 98.51% and 48.99%, respectively. The expression levels of TES, TES (1), PksA and PksJ were significantly down-regulated by 89.02%, 98.20%, 46.49% and 40.13%, respectively. However, the content of non-host selective toxin ATX- I of ABL2 strain and the expression of PksF gene involved in its synthesis increased.【Conclusion】The SC5 metabolites of T. longibrachiatum at a concentration of 0.5 mg·mL^-1 had an inhibitory effect on the growth and pathogenicity of A. tenuissima ABL2 strain. It could reduce and inhibit the content of TEN, ALT and TeA by down-regulating the expression of TES, TES (1), PksA and PksJ genes in ABL2 strain, thereby reducing its pathogenicity. This study can provide a theoretical basis for the biological control of ABL2 strain.