| In this study, Fusarium oxysporum f.sp. cubense race4 (FOC4) was used as a plant pathogen, and Trichoderma was the biocontrol fungi which was isolated and purified from the laboratory. Select Trichoderma strain with better biocontrol effect, and determine the composition and content of volatile compounds of Trichoderma and its effect on FOC4 hyphae and respiration. The antagonistic mechanism of volatile substances produced by Trichoderma strain to FOC4 was preliminarily explored. The results are as follows:1. The results of confrontation experiment showed that the growth rate of 426 strains of Trichoderma was much higher than that of FOC4. Trichoderma and FOC4 showed four different plate characteristics: (1) Trichoderma mycelium was covered with FOC4 colonies and produced a large number of conidia; (2) Trichoderma mycelium was covered with FOC4 colonies did not produced spores, but the FOC4 colony turned into yellow; (3) Two strains appeared noticeable white mycelium at the contact ring; (4) A yellow band appeared at the contact of the two strains. The results showed that 426 strains of Trichoderma spp. had different inhibitory effects on FOC4, among which there were 7 strains with more than 80%antifungal rate, the highest inhibitory rate of strain 225-2P1 was 83.33%, and the antibacterial rate of strain 79-1P2 was the lowest, which was 50.00%.2. The results of pair test experiment showed that all tested Trichoderma was growed normally when inoculated on PDA plates, but the growth rate of FOC4 on PDA plates was gradually slowed down. Trichoderma volatile compounds and FOC4 showed four different plate characteristics: (1) Colony diameter becomed smaller; (2) Colony diameter becomes smaller and mycelium becomed thinner; (3) Colony diameter becomed smaller and hyphae deformation. For the 426 strains of Trichoderma strains volatile substances on FOC4 had different degrees of inhibition, antifungal rates were less than 40%, which inhibition rate of more than 30% of a total of 11 strains, the highest inhibitory rate of strain 225-2P1 was 37.18%, followed by strain 223-2M1, which was 35.29%.3. According to the results of confrontation and the pair test experiment, 21 strains with good inhibitory effect were screened, and 21 strains of Trichoderma were identified by morphological and ITS sequence analysis. The results showed that 21 strains of Trichoderma were divided into three species, namely: T. harzianum 8 strains, numbered 71-1P2, 72-1P4,173-1P2,209-1P1,223-2M1,226-2P1,233-1P1,250-1P1; T. atroviride 11 strains,numbered 5-1P1, 16-1M1, 16-1M2, 70-1M3, 193-2P1, 222-2M1,225-2P1, 231-1M2, 233-2M1,247-2P2, 258-2M1; T. longibrachiatum 2 strains,numbered 205-2P1 and 221-2M1.4. The different types and contents of volatile substances produced by different Trichoderma strains were different. A total of 434 kinds of volatile substances were detected in 21 strains of Trichoderma, including 99 kinds of alkanes, 78 kinds of esters, 69 kinds of olefins, 45 kinds of alcohols, 40 kinds of ketones, 12 kinds of benzene, 12 kinds of ethers,the other 79 kinds, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane two compounds can detected in 21 strains of Trichoderma. Cluster analysis in the correlation coefficient distance of 8, 21 strains of Trichoderma strains can be classified as four. 434 kinds of volatile substances can be simplified as 9 main components, including 30 kinds of volatile substances. Different Trichoderma volatile compounds of the main component of the score and antagonism of FOC4 between the inhibition rate is not significant, but the general trend of the two is consistent, indicating that the species of Trichoderma volatile substances and the inhibition rate of FOC4 have a certain relationship.5.Trichoderma volatile substances can inhibit the growth of FOC4, 16-1M1,225-2P1,233-1P1,233-2M1,250-1P1 these five strains of the effect is better, of which 16-1M1 on FOC4 the inhibition rate of mycelial growth was the highest, 48.97%, the sporulation rate of 250-1P1 strain was 98.97%, the spore germination rate of 225-2P1 strain was the lowest,was 20.02%. Trichoderma spp. showed that the mycelial morphology of FOC4 showing five characteristics: (1) The branches were reduced, the mycelial thickness was uneven and the twist was distorted; (2) The hyphae were dissolved at the junction; (3) Mycelium wound into a ball, and some will appear in the phenomenon of conidia buried; (4) Cell contents of the extravasation, adhesion to the mycelium surface; (5) Hyphae Surface serious damage, the top of the expansion, and some holes.6.When the medium contains different concentrations of (0,1,5μg/mL) ATP, the inhibitory effect of Trichoderma viride on FOC4 decreased with the increase of ATP concentration. The respiration rate of FOC4 hyphae was higher than that of the control value,that is, the volatile matter of Trichoderma spp. could promote the respiration of FOC4. The effect of strain 250-1P1 was the most obvious, followed by strain 233-2M1, 233-1P1. By adding a typical inhibitor of different metabolic pathways, it can be found that the volatile substances of Trichoderma spp. Mainly promote the respiration of FOC4 through the glycolysis pathway, followed by the tricarboxylic acid pathway. Trichoderma volatile compounds can inhibit the activity of NADH-Q reductase and alternate oxidase, which in turn affects mitochondrial respiration electron transport,inhibit ATP synthesis, resulting in insufficient energy supply, thus limiting the growth of FOC4. |
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