关键词: 腐霉菌, 茎腐病, 玉米, 非洲哈茨木霉, 生物防治

Abstract:

【Objective】The objective of this study is to screen Trichoderma strains which have inhibitory effect on the Pythium spp. causing maize stalk rot, and to clarify their taxonomic status, control efficacy and antifungal mechanism. This study will provide important resources for the research and development of biocontrol agent against Pythium stalk rot.【Method】For the antagonistic strains screening, the inhibitory effect of Tichoderma strains on P. inflatum, P. arrhenomanes and P. aristosporum was tested by measuring the mycelia growth. The taxonomic status of Tr21 was determined by morphological and molecular characteristics. The effect of Tr21 on the mycelia morphology of Pythium spp. was observed in the laboratory. In order to analyze the effect of Tr21 fermentation broth on the membrane permeability of Pythium spp., propyridine bromide (PI) dye solution was used to stain, and the absorbance values of protein and nucleic acid in mycelia supernatant at different treatment times were detected. The effect of Tr21 fermentation broth on germination characteristics of maize seeds was tested by seed soaking with different concentrations of fermentation broth. The control efficacy of Tr21 on stalk rot was confirmed through greenhouse pot and field inoculation experiments.【Result】From the 109 strains of Trichoderma spp., seven strains were screened with antagonistic activity against P. inflatum, P. arrhenomanes and P. aristosporum, and the inhibition rate was above 60%. The inhibition rate of Tr21 to three Pythium species reached 100%, the inhibition rate of 5×, 10× and 20× diluent to three Pythium species reached 100%, and the inhibition rate of 50× diluent to three Pythium species was also more than 55.56%. Tr21 strain was identified by morphological and molecular biology as T. afroharzianum. The results of microscopic observation showed that the fermentation broth of Tr21 could cause mycelial malformations, such as rough mycelia, increased mycelial branching, shortened nodes, and overflow of mycelia contents. The result of PI fluorescence stain showed that the cell membrane of three Pythium species was damaged by Tr21 fermentation broth, and the PI dye was more likely to penetrate the damaged cell membrane into the mycelium and stain the mycelia red. The results of nucleic acid and protein leakage showed that the absorbance values of the mycelia treated by the fermentation broth changed greatly. After treatment for 5 h, the OD₂₆₀ increased by 0.08 and OD₂₈₀ increased by 0.10, 0.11 and 0.10, respectively, indicating that the membrane of the mycelia was damaged, leading to the overflow of mycelia contents. The different concentrations of Tr21 fermentation broth had no effect on the germination characteristics of maize seeds, and the 20× diluent had the best effect on germination and growth of seeds. The results of pot experiment showed that 5× diluted fermentation broth of Tr21 had the best control efficacy on Pythium stalk rot caused by three Pythium species, which was 60.67%, 63.15% and 59.66%, respectively. The control efficacy on Pythium stalk rot of 5× diluent was the highest, reaching 82.25%, with a mass ratio of 1﹕100 (5× diluent to seed).【Conclusion】An effective T. afroharzianum strain Tr21 was obtained for preventing and controlling of maize Pythium stalk rot. The fermentation broth of Tr21 can lead to mycelia malformation, breakage, cell membrane damage and contents leakage, etc. In conclusion, the T. afroharzianum strain Tr21 is a promising biocontrol microbial.

Key words: Pythium, stalk rot, maize, Trichoderma afroharzianum, biological control