Production of Microsclerotia by Metarhizium sp., and Factors Affecting Their Survival, Germination, and Conidial Yield

Abstract

Microsclerotia (MS) produced by some species of Metarhizium can be used as active ingredients in mycoinsecticides for the control of soil-dwelling stages of geophilic pests. In this study, the MS production potential of two Metarhizium brunneum strains and one M. robertsii strain was evaluated. The three strains were able to produce MS in liquid fermentation, yielding between 4.0 × 10⁶ (M. robertsii EAMa 01/158-Su strain) and 1.0 × 10⁷ (M. brunneum EAMa 01/58-Su strain) infective propagules (CFU) per gram of MS. The EAMa 01/58-Su strain was selected for further investigation into the effects of key abiotic factors on their survival and conidial yield. The MS were demonstrated to be stable at different storage temperatures (-80, -18, and 4 °C), with a shelf-life up to one year. The best temperature for MS storage was -80 °C, ensuring good viability of MS for up to one year (4.9 × 10¹⁰ CFU/g MS). Moreover, soil texture significantly affected CFU production by MS; sandy soils were the best driver of infective propagule production. Finally, the best combination of soil temperature and humidity for MS germination was 22.7 °C and 7.3% (wt./wt.), with no significant effect of UV-B exposure time on MS viability. These results provide key insights into the handling and storage of MS, and for decision making on MS dosage and timing of application.

Keywords: UV-B; entomopathogenic fungi; moisture; resistance structures; soil texture; temperature.

Figure 1

Microsclerotia of Metarhizium sp. observed after 10 days in liquid fermentation at 28 °C and 300 rpm: (A) microsclerotium of M. robertsii EAMa 01/158-Su strain; (B) microsclerotium of M. brunneum EAMb 09/01-Su strain; (C) microsclerotium of M. brunneum EAMa 01/58-Su strain. The photomicrograph was taken at 100× magnification, using an optical microscope (Motic BA400, Barcelona, Spain) and a digital camera (Leica DFC 450, Barcelona, Spain).

Figure 2

(A) Microsclerotia production in liquid medium after 8 days at 25 °C; (B) CFU produced per gram of microsclerotia. Mean values (±SE) followed by different letters are significantly different to one another according to Fisher’s protected Tukey’s HSD test (p ≤ 0.05).

Figure 3

Conidia yield per gram of M. brunneum EAMa 01/58-Su strain microsclerotia after 4, 8, and 12 months at −80 °C, −18 °C, 4 °C, and 25 °C. Mean values (±SE) within temperature followed by different letters are significantly different to one another according to the mean separation test with 95% protected by Tukey’s HSD range test (p ≤ 0.05).

Figure 4

Time course of CFU yield (mean ± SE) of M. brunneum strain EAMa 01/58-Su microsclerotia as a function of incubation time.

Figure 5

Time course of CFU yield per gram of M. brunneum strain EAMa 01/58-Su microsclerotia, according to the type of soil, over a period of 3 months. Mean values (±SE) within the evaluation period (days) followed by different letters are significantly different to one another according to Fisher’s protected Tukey’s HSD test (p ≤ 0.05).

Figure 6

Effects of temperature and moisture on the CFU yield per gram of M. brunneum strain EAMa 01/58-Su microsclerotia. The mesh plots represent the predicted values, and the dots represent actual data.