Elsevier

Fungal Biology

Volume 122, Issue 9, September 2018, Pages 883-890
Fungal Biology

Modified Adamek's medium renders high yields of Metarhizium robertsii blastospores that are desiccation tolerant and infective to cattle-tick larvae

https://doi.org/10.1016/j.funbio.2018.05.004Get rights and content

Abstract

Blastospores are yeast-like cells produced by entomopathogenic fungi that are infective to arthropods. The economical feasible production of blastospores of the insect killing fungus Metarhizium spp. must be optimized to increase yields. Moreover, stabilization process is imperative for blastospore formulation as a final product. In this sense, our goal was to increase blastospore production of two Metarhizium isolates (ESALQ1426 and ESALQ4676) in submerged liquid cultures. A modified Adamek's medium was supplemented with increased glucose concentrations and the fermentation time was accelerated by using a blastospore pre-culture as inoculum. Virulence of air-dried stable blastospores was compared with conidia toward larvae of the cattle tick, Rhipicephalus microplus. Our results revealed that blastospore production of Metarhizium is isolate- and species-dependent. Glucose-enriched cultures (140 g glucose/L) inoculated with pre-cultures improved yields with optimal growth conditions attained for Metarhizium robertsii ESALQ1426 that rendered as high as 5.9 × 108 blastospores/mL within 2 d. Resultant air-dried blastospores of ESALQ1426 were firstly proved to infect and quickly kill cattle tick larvae with comparable efficiency to conidia. Altogether, we argue that both osmotic pressure, induced by high glucose titers, and isolate selection are critical to produce high yields of blastospores that hold promise to control cattle-tick larvae.

Introduction

Metarhizium spp. (Ascomycota: Hypocreales: Clavicipitaceae) are cosmopolitan fungi used as biological control agents against arthropod pests in agriculture, livestock and vectors of human diseases (Alkhaibari et al., 2016, Camargo et al., 2016, Lee et al., 2015, Van Lenteren et al., 2017). Interestingly, asexual spores termed conidia of Metarhizium anisopliae have been applied in Brazil on more than 3 million hectares of sugarcane annually for controlling spittlebugs (Hemiptera: Cercopidae) (Mascarin et al., 2018, Parra, 2014). This is indeed one of the most successful biological control programs ever practiced using a single fungal entomopathogen in the world.

Nonetheless, besides conidia, other fungal infective structures have been investigated for use in biological control programs. One such type of propagule is a yeast-like cell termed blastospore. Blastospores are formed in the arthropod hemolymph during fungus infection. When applied against some hosts, earlier reports document that blastospores can be even more virulent than conidia (Alkhaibari et al., 2016, Alkhaibari et al., 2017, Jackson et al., 1997, Kim et al., 2013, Mascarin et al., 2015a, Mascarin et al., 2015b, Mascarin et al., 2016, Wassermann et al., 2016); moreover, the process for blastospore production has a number of advantages over that for conidia production (Jackson et al., 1997; Jaronski and Mascarin, 2013).

Large amounts of blastospores (>108 blastospores/mL) can be achieved by liquid culture fermentation in a shorter time (less than 4 d), in a smaller space and with lower labor requirement, than the solid-substrate fermentation method used for conidia production. Furthermore, the liquid fermentation method has a standardized quality similar to that found in commercial yeast production, which facilitates its scale-up production and downstream processing. In spite of clear advantages in the system production of blastospores relatively to that of conidia, the former is highly vulnerable to environmental stress conditions (Jaronski and Mascarin, 2013).

Therefore, to develop a blastospore biopesticide, it is necessary to devise formulations that afford good stability and protection against deleterious environmental factors. With this in mind, one of the key conditions for developing a blastospore biopesticide arises from the stabilization process to tolerate drying. Stabilization is necessary to decrease the blastospore metabolism while keeping it viable for long periods. Once stabilized, blastospores can be formulated aiming at shelf life extension and meeting the requirements for commercialization (Jackson et al., 1997, Kim et al., 2013, Mascarin et al., 2016).

There are several technologies used for cell stabilization but one of the most feasible and economically viable is air-drying, which consists of a slow drying process where the relative humidity of the air flow is controlled throughout the course of drying in order to maintain cell integrity (Jackson and Payne, 2007, Mascarin et al., 2016). Blastospores of some fungi can resist the air-drying process and remain highly viable for up to 12 m (Mascarin et al., 2015a, Mascarin et al., 2016). Some studies with the entomopathogenic fungi of the genera Beauveria and Isaria showed that stabilized blastospores can be significantly more virulent than conidia toward some hosts (Jackson et al., 1997, Mascarin et al., 2015a, Mascarin et al., 2015b, Mascarin et al., 2016, Vandenberg et al., 1998). However, this outcome is highly dependent on the fungal isolate and on the medium composition.

The standard Adamek's medium has been successfully used to produce Metarhizium blastospores although the yields rarely reach high concentrations (>108 blastospores/mL). Recently Adamek's medium has been shown to support the infectivity of fresh blastospores produced against the larvae of mosquitoes (Alkhaibari et al., 2016, Alkhaibari et al., 2017) and of Ixodes ricinus ticks (Wassermann et al., 2016).

The cattle-tick, Rhipicephalus microplus Canestrini (Acari: Ixodidae), is a one-host ectoparasite of livestock that inhabits mainly the tropical and subtropical regions of the world (Estrada-Peña et al., 2006). This ectoparasite is a vector of important pathogens: the bacterium Anaplasma marginale, and the protozoa Babesia bigemina and B. bovis Babes, responsible for causing the bovine diseases anaplasmosis and babesiosis, which results in an economic loss of US$3.2 billion per year (Grisi et al., 2014) by dramatically reducing weight gain of cows, milk production, the price of leather and the survival of calves.

Cattle-ticks have hitherto been prophylactically controlled with synthetic acaricides applied mainly as a pour-on directly on cows. However, concerns about the residues of acaricides in milk, the increasing threat posed by acaricide resistance in tick populations (Abbas et al., 2014, Mendes et al., 2013) and the lack of other alternatives to cattle-tick control have highlighted biological control as a promising alternative for controlling ticks in the field.

It is estimated that only 10–20 % of cattle-ticks are actually in their hosts, whereas the remaining 80–90 % are in the non-parasitic phase in the field, a phase mainly represented by larvae (Leal et al., 2017) that can survive long periods without feeding. Considering that each engorged female lays 2000 to 4500 eggs on the ground (Wall and Shearer, 2001) and that larvae can survive for long periods, spraying fungal infective structures on grasslands is a potential alternative for integrated management of cattle-tick.

The efficiency of Metarhizium spp. conidia against ticks under field conditions has been well reported in the literature (Camargo et al., 2016, Murigu et al., 2016, Ojeda-Chi et al., 2010, Samish et al., 2014). However, the effective control of cattle ticks requires high concentrations of conidia (up to 1 × 109 conidia/mL), sprayed over extensive grassland areas (Fernandes et al., 2011), which may discourage the use of fungal entomopathogens due to the high cost of spraying.

Taking into account that liquid culture fermentation is relatively quicker as well as more cost-effective than solid-state fermentation and that blastospores can potentially replace conidia for tick control, the aim of this work was to i) Select Metarhizium isolates that achieve a high yield of blastospores, ii) Improve Adamek's medium for the production of high concentrations of blastospores in short fermentation times; iii) Assess efficiency and efficacy against Rmicroplus larvae of air-dried blastospores of the Metarhizium isolate that showed the highest blastospore yields in the improved medium in comparison with that of conidia.

Section snippets

Isolate selection

Eleven Metarhizium spp. isolates were chosen from the Entomopathogenic Fungal Collection from ESALQ-University of São Paulo (Piracicaba, Brazil). We selected isolates from M. anisopliae, Metarhizium robertsii, Metarhizium brunneum, and from two species not phylogenetically determined, recovered from different Brazilian states, referred to as Metarhizium sp. undet 1, Metarhizium sp. undet 4. The selected isolates for each species were: M. anisopliae: ESALQ2787, ESALQ4676 and ESALQ1184, M.

Screening isolates for blastospore production in Adamek's medium

There was a remarkable variation in blastospore concentration of fungal isolates of different Metarhizium species at 2, 3 and 4 d of culture, indicating that these fungal isolates exhibited distinct growth rates (F = 4.05, df = 20, 155, P < 0.0001) (Fig. 1). We noted that two isolates of M. robertsii (ESALQ1426 and ESALQ4130) were the best producers attaining concentrations up to 3.6–3.8 × 108 blastospores/mL by day 4. These isolates also had the quickest growth rates as blastospore yields

Discussion

The present work highlights for the first time the infectivity of stabilized air-dried blastospores of M. robertsii (ESALQ1426) against cattle-tick larvae. Tolerance to stabilization processes is known to vary according to the species and/or isolate and the growing medium. Thus, we showed that blastospores produced by isolate ESALQ1426, grown in modified Adamek's medium with salts, vitamins, trace metals and high glucose concentrations (140 g/L) survive well to the air-drying stabilization

Conflicts of interest

The authors declare no conflict of interest.

Author contributions

NS and ID planned the research and designed the experiments. NS and BO performed the experiments. GM analyzed data. NS, ID and GM wrote the manuscript. All authors discussed results and commented on the manuscript.

Acknowledgments

This work was funded by, The Brazilian National Council for Scientific and Technological Development (Cnpq) [grant number 421629/2016-9]. The first author thanks the São Paulo Research Foundation (FAPESP) [grant number 2016/20610-6] and the Coordination for the Improvement of Higher Educational Personnel (CAPES), for scholarships during the PhD project. We thank E. Potworowski for critically reviewing an earlier draft of this manuscript.

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