Enzymatic activity and virulence of Cordyceps locustiphila (Hypocreales: Cordycipitaceae) on the South American locust Schistocerca cancellata (Orthoptera: Acrididae)

2.1 Fungal isolation

The entomopathogenic C. locustiphila was found infecting T. collaris, in an undisturbed area of the Yabotí biosphere reserve (26° 37′ S 53° 40′ W), a naturally protected region that includes part of the departments of Guaraní and San Pedro in Misiones province, Argentina. The grasshopper T. collaris colonized by C. locustiphila was collected in a plastic recipient with silica gel and posteriorly deposited at the Herbarium of the Spegazzini Institute of Botany, La Plata National University (access code LPS 49245). The entomopathogenic anamorph was also isolated from T. collaris and deposited at the Spegazzini-Institute–culture-collection (access number LPSc 1218) and PCR products obtained were sequenced and deposited at GenBank (accession numbers: MF185185; MF185186; MF185187; MF185188).

2.2 Pathogenicity assays

The C. locustiphila anamorph was cultured on potato-dextrose-agar medium for 10 days in the dark at 25 °C to promote conidia production. Posteriorly, conidia were collected (Fig. 1) with disposable cell scrapers (Fisherbrand™) and put into test tubes containing 0.01% (v/v) Tween 80™ (polyoxyethylene sorbitan monolaurate) (Merck). The resulting suspensions were vortexed for 2 min and subsequently filtered through four layers of sterile muslin. The concentration of solutions was adjusted to 1 × 10⁴, 1 × 10⁶ and 1 × 10⁸ conidia/ml using a Neubauer haemocytometer (Marienfeld™; Germany) and conidial viability was established for each stock suspension according to Goettel and Inglis (1997). The average viability of the conidia was in all cases over 95%.

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Fig. 1

Electron microscopy photo of conidia the anamorphic form of Cordyceps locustiphila.

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Third-instar S. cancellata nymphs were used for pathogenicity assay. Treatments consisted of different concentration of conidial suspensions (1 × 10⁴, 1 × 10⁶ and 1 × 10⁸ conidia/ml). Three replicates of 10 individuals (on different dates) were sprayed with 1 ml of each treatment using a 35 ml glass atomizer. For each replicate and treatment, 10 control insects were atomized with 1 ml of 0.01% [v/v] Tween 80™ only. Treated and control locusts were placed in 50 × 9-cm acetate tubes with screened ends (Henry, 1985) and fed with lettuce, cabbage, and wheat bran. The acetate tubes were maintained in climatic chambers under controlled conditions (30 °C, 60% relative humidity and 14:10-h light:dark photoperiod). The cumulative mortality was recorded daily for 10 days. Mycosis was confirmed by examination of dead locusts under the microscope (ZEISS-Primus Star-Germany).

2.3 Production of enzymes at different temperatures on solid media

The lipolytic, proteolytic, amylolytic, cellulolytic, and chitinolytic activities were studied performing a plate test in agar media with specific substrates: Tween 20™, casein, starch, carboxymethylcellulose, and Chitin Azure, respectively (Hankin and Anagnostakis, 1975). The C. locustiphila strain was cultivated in Petri dishes containing malt extract agar at 26 °C, for seven days in the dark to obtain agar plugs (7 mm diameter) from the fungal active growth zone, which acted as inocula. Inocula was placed on the centre of 9 mm Petri dishes containing agar media and the different substrates to estimate lipolytic, proteolytic, amylolytic, cellulolytic, and chitinolytic of the funguns. Petri dishes were incubated in growing stove (Semedic I291PF) at 4 ± 1 °C, 15 ± 1 °C and 26 ± 1 °C for 15 days (Kathiresan and Manivannan, 2006; Saparrat et al., 2008; Sabu et al., 2012; Sudarkodi et al., 2015). The existence of a halo surrounding the fungal colony was recorded to estimate enzymatic activity. A total of three repetitions were performed for each treatment (enzymatic activity and temperature).

An opaque halo around the colony due to the precipitation of the calcium salt of the fatty acid was present when lipolytic activity was occurring, proteolytic activity was visualized as a transparent halo, a consequence of the degradation of casein (Koneman and Roberts, 1987), and the chitinolytic activity also was revealed when a transparent halo formed as Chitin Azure was depolymerized by the activity of the fungus (Howard et al., 2003). Amylolytic and cellulolytic activities were evident when a transparent enzyme degradation halo formed as well. To determine amylolytic activity, a 1% iodine solution in 0.2% KI solution (lugol) was used (Hankin and Anagnostakis, 1975). The cellulolytic activity was revealed with 0.2% Congo Red 5% acetic acid, and 1 M sodium chloride.

2.4 Production of enzymes in liquid medium

A Sabouraud dextrose liquid medium (150 ml) containing 0.5% (w/v) peptone meat, 0.5% (w/v) casein peptone, and 2% (w/v) glucose was prepared and four malt extract agar (MEA) plugs of C. locustiphila culture were incorporated. These were cultivated for eight days at 30 ± 1 °C, darkness and in orbital shaker (Labdiscount™) at 180 rpm. This constituted the crude extract for further determination of enzymatic activity (Mancillas-Paredes et al., 2019).

2.5 Enzyme extracts

The chitinolytic, keratinolytic, and caseinolytic activities were determined using the crude extract produced by C. locustiphila. The extract was filtered by vacuum pressure using OSMONICS borosilicate fiberglass discs (0.45 µm) and kept at 4 °C. To establish chitinolytic activity methods by Kuzu et al. (2012) with modifications were carried out. Briefly, 0.003 g of Chitin Azure was suspended in 0.6 ml of Tris-HCl buffer (0.1 M and pH 8) and then incorporated to 0.6 ml of the crude extract. This solution was incubated at 28 °C for 2 h and posteriorly the reaction was stopped in a cold bath and centrifuged (5000 g) for 15 min at 5 °C. The blanks were obtained by heat inactivation of the crude enzyme extract and the absorbance established at 595 nm. The absorbance variation per time unit was measured and one unit (U) of enzyme activity was defined as the amount of enzyme required to vary 0.01 absorbance in a minute, at 31 °C and pH 8. The proteolytic activity was determined using casein at 1% (w/v) in 0.1 M Tris-HCl buffer, pH 8.0 that was heated in a water bath for 20 min at 35 °C to accomplish its dissolution. The reaction mixture contained 50 µl of crude extract and 550 ml of casein. The reaction time was 30 min and stopped afterwards with 90 µl of TCA 5% (w/v). The blanks consisted of the same reaction mixture, but changing the order in which components were added: 1) crude extract, 2) TCA 5% and 3) casein. After stopping reactions, products were maintained for 15 min at 4 °C and posteriorly centrifuged at 4800g for 20 min at 25 °C (López et al., 2000). Absorbance was measured at 280 nm considering a caseinolytic unit (Ucas) as the amount of enzyme required to produce an increase of one absorbance unit at 280 nm per min. Finally, a mix containing 150 μl of filtered enzyme extract per 0.22 μm and 250 μl of a 1% w / v solution of azocasein (Sigma Chem. Co.) in 0.1 M Tris-HCl buffer, pH9 was prepared in order to establish the azocaseinolytic activity of the fungus. The solution was incubated for 30 min at 37 °C and the reaction stopped by adding 1 ml of 10% w/v trichloroacetic acid. The product was centrifuged at 3000g for 15 min and 0.9 ml of the supernatant mixed with 1 ml of 1 N NaOH. The spectrophotometric lectures were made at 440 nm. The crude extract was heated at 100 °C for 5 min to inactivate enzymatic activity and used as reaction blank. The amount of enzyme that caused an increase of 0.1 Abs per minute under the reaction conditions was considered the azocaseinolytic unit.

2.6 Statistical analysis

The Trimmed Spearman Karber analysis was performed to estimate LC50 of the entomopathogen with 95% confidence limits (CL). Data on the enzymatic activity of C. locustiphila at the different temperatures were analysed using a two-way ANOVA. Analyzes of variance were performed with the InfoStat 2007 software (InfoStat, 2001).