| Summary: | Entomopathogenic fungi (EPF) are regarded as viable alternatives to insect pest control chemicals that contain a large amount of protease enzyme, which degrades the proteinaceous substances in insect cuticles. The aim of this study was to evaluate the field efficacy of protease-producing EPF against <i>Ceratovacuna lanigera</i>, and to assess the persistence of the <i>Ceratovacuna lanigera</i>, on sugarcane leaves. A total of 10 protease-producing fungi isolates were cultured from different agricultural soils, and identified as <i>Purpureocillium lilacinum</i> on the basis of the morphological features and molecular data, using ITS 1-5.8 S-ITS 2 of the rDNA sequences. The proteolytic activity of the isolates was assessed and expressed as an Enzyme Index (EI). Three isolates (PLTP5, PLPS8, and PLMC11) were found to be the best enzyme producers among the isolates, presenting EI values > 2.0 at 24 h, 48 h, and 72 h during incubation. These three isolates also gave the best results in terms of lethal concentrations (LC<sub>50</sub> and LC<sub>90</sub>) and lethal time (LT<sub>50</sub> and LT<sub>90</sub>) values, based on laboratory virulence evaluation, and were therefore selected for field application; commercial <i>B. bassiana</i> (GHA) was applied as standard treatment. An oil-based formulation of conidia (1 × 10<sup>8</sup> mL<sup>−1</sup>) of the isolates was applied in a sugarcane field experiment, with an interval of 10 days for four times. The results revealed that <i>P. lilacinum</i> (PLTP5) showed the highest reduction in the <i>C. lanigera</i> population, of 28.75, 56.02, 67.42, and 77.21%, respectively, after the first, second, third, and fourth spraying. The persistence of the conidia of the isolates on the sugarcane leaves was assessed. Per cent reductions in the conidia population, of 36.94–45.23%, 69.36–80.15%, and 81.75–92.96%, respectively, were found after three, six, and nine days of application. The application impact of EPF on the yield component and sugar content was evaluated. <i>Purpureocillium lilacinum</i> (PLTP5) showed the highest increase, of 18.15, 17.83, 15.07, 11.88, 23.73, and 19.38%, respectively, for leaf length, leaf width, cane height, cane girth, cane weight, and sugar content (brix). Our study indicated that <i>P. lilacinum</i> (PLTP5) was the most effective EPF against <i>C. lanigera</i> in field conditions, and also showed the highest proteolytic activity. Consequently, protease was considered the essential factor regulating the efficacy of <i>P. lilacinum</i> against <i>C. lanigera</i>. Protease would be useful, therefore, as an eco-friendly alternative to chemical pesticides, for the sustainable management of <i>C. lanigera.</i>
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