【Background】 Nimbya alternantherae has been considered an important biocontrol agent of Alternanthera philoxeroides. The capacity of conidia production and pathogenicity of the fungus had been reduced because of subculture or preservation for long periods in the lab, causing potential problems for its use in biological control projects. 【Method】The rejuvenations of N.alternanthera SF193, first and second generations were obtained by the process of inoculation, isolation, purification and identification. Conidial production of the initial and the rejuvenated strain was investigated after conidial cultivation for 3, 6, 9 days. The efficiency of A.philoxeroides treated by the initial and the rejuvenated strains was evaluated in the field. 【Result】Ten rejuvenated first generations and ten second generation isolates were obtained. All of the rejuvenated isolates could cause foliar and stem necrosis of A.philoxeroides and the morphological character of conidia of rejuvenated isolates were the same as that of the initial isolate. Compared to the initial isolate, the conidial yield of rejuvenated isolate increased remarkably, and the time to peak conidial yield of rejuvenated isolate was shortened sharply. The rejuvenated second generation showed the highest conidial yield and was about 4.[KG-*8]8 times more likely to produce conidia than the initial isolate. The times to peak conidial yield between the initial and rejuvenated isolates were 3 and 6 days, respectively. Under field conditions, the level of disease severity of A.philoxeroides by myceliablended suspensions of first generations and second generations without dilution ware increased by 4.[KG-*8]65% and 9.[KG-*8]82% and also by 25.[KG-*8]79% and 16.[KG-*8]55% after myceliablended suspensions diluted 1∶[KG-*2]10 (V/V) with water, compared to the original isolate. 【Conclusion and significance】It is important to sustain the genetic stability of N.alternanthera SF193 and improve the efficiency of biocontrol isolate against the weed by periodically rejuvenating the fungal isolates which were subcultured or preserved for long periods in the laboratory.