Potential Use of Species in the Erynioideae for Biological Control and Biotechnology

1. Introduction

The fungal order Entomophthorales in the Zoopagomycotina includes at least 246 species of arthropod pathogens [1], many of which are well known for the ability to cause epizootics and change the behavior of infected hosts [2]. Their role in biocenoses is extremely important because they can function as regulators of arthropod populations and thus play a role in ecosystem homeostasis. Within the Entomophthorales, the largest family is the Entomophthoraceae, which consists exclusively of arthropod pathogens. This family was divided into two subfamilies in 2005, the Entomophthoroideae and the Erynioideae [3]. The Erynioideae is the larger of these two subfamilies, containing five genera, namely Erynia, Furia, Pandora, Strongwellsea, and Zoophthora. Fungi in these genera use their ballistic conidia to infect a wide range of arthropod species inhabiting agricultural and forest ecosystems, including insect hosts that are recognized as pests of various important crops worldwide [4,5]. In addition to attacking arthropod pests directly damaging crops and forests, directly some of the arthropods hosts of species in this subfamily include vectors of numerous diseases that impact humans, livestock, and crops.

Due to the observed capability of the Entomophthorales to cause massive mortality of insect hosts, questions about their potential use for various biotechnological applications are often raised. However, with the exception of the experimental release of Zoophthora radicans in Australia in the late 1970s [6] no other species of Entomophthorales have been developed thus far for field testing as fungal biological control agents. Species within this group differ from one another in numerous ways that impact their potential development as biopesticides [7]. One of the most important attributes to consider in this regard is the cultivability of these fungal species in vitro, which plays a critical factor in their propagation for potential application as biological control agents. This feature can be expressed to various degrees: from total inability to isolate fungal strains in vitro to routine transfers of the isolates and preservation of their cultures in culture collections, to research on improving in vitro growth toward potential mass production. Additionally, for successful biological control, the host range of the pathogens must be known, and is crucial in both identifying suitable fungi for specific target pests as well as in avoiding potential impacts on non-target arthropods. Furthermore, the natural habitats of these fungi and their geographical distributions are important toward consideration of development for biological control as regions around the world differ in regulation of species to be used for pest control that are native vs. non-native [8]. In addition, knowledge of the habitats where these fungi are naturally active will provide information about their ecological adaptability and long-term survival under diverse conditions.

We have analyzed these vital characteristics for species of the Erynioideae, and we aim to pinpoint which species demonstrate the most potential for future use in biological control. Moreover, we are keen on identifying the specific insect groups for which the application of these species as biological control agents might be most successful. We predict that these findings can be used as bases for screening and selection in future biocontrol research programs with Entomophthorales, paving the way for more effective and sustainable pest management solutions with species in the Erynioideae.

The primary objective of this research was to characterize several critical aspects of the lifestyles of the species within the five genera in the Erynioideae, here referred by the acronym EFPSZ. An overall consideration of these aspects of species in this group has not previously been undertaken. We predict that the assembly of this new information could be important toward potential applications of this group in various aspects of biotechnology, particularly in the development of biological control agents for pest management.

2. Materials and Methods

2.1. Literature analysis

We sought all literature related to the five genera in the Erynioideae through the use of the Web of Science, Scopus, and Google Scholar. We also examined the information associated with the species and strains deposited in the US Department of Agriculture Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF), the American Type Culture Collection (ATCC, USA) and CBS-Westerdijk Institute KNAW Fungal Biodiversity Centre, also known as Central Bureau of Fungal Cultures (Netherlands). The traits that were investigated are geographical distribution, host range, type of habitat, and documented ability to grow in vitro.

2.2. Distribution map

A map of the number of recorded species was created using StepMap GmbH software (Berlin, Germany). Countries and regions were colored according to the number of recorded species from light (less than five species) to dark pink (over 50 species described). Green indicates none have been reported. We consider the species: 1) local if the distribution range covers only one continent, 2) cosmopolitan or broadly distributed with records on at least two continents, and 3) ubiquitous if distribution records cover three or more continents.

2.3. Phylogenetic tree

To generate a dataset of EFPSZ taxa, we downloaded 18S and 28S sequences of identified species with accurate nomenclature from GenBank. All sequences were initially aligned, their alignments were manually adjusted, and ambiguous regions were excluded from the alignments using Mesquite 3.04 version [9]. Phylogenetic relationships were determined by the Neighbor Joining (NJ) algorithm, and the tree was visualized in PAUP* 4.0 [10].

3. Results

3.1. Geographic distribution

Species in the EFPSZ group have been recorded from all continents except Antarctica. However, the number of described species differs significantly between countries and regions (Figure 1). Many records are from several Central European countries (especially Switzerland, Poland), and the United Kingdom. Many species have also been found in North America (USA and Canada), other European countries, and China. Only a handful of records have been made in South America and across a considerable area of Asia and Oceania. The continent least documented for these fungi is Africa, where EFPSZ fungi have been reported only from four countries. They are also sparsely reported in countries in South America other than Argentina, Brazil, and Chile and in several countries in the Middle East. However, the map in Figure 1 most likely does not represent the actual species distributions, but rather the situation regarding our knowledge of this fungal group in particular countries where more sampling has occurred. It is obvious that climatic conditions in many countries of Africa or South America might be very favorable for species in the Erynioideae, but little research has yet been undertaken to describe Erynioideae and their host ranges from these regions.

There were clear differences in pattern species distribution among the five genera. Each genus contains both ubiquitous and cosmopolitan species as well as local ones, and they are not grouped in any specific way on the phylogenetic tree (Figure 2). It is very possible that many of the species of these genera that we classify as local have much broader distributions but have not been sampled broadly. We consider the broad distributions of many species as advantageous for future biocontrol agents since this feature might indicate a significant level of adaptability and ability to survive the environmental conditions in different climatic environments due to their ecology, pathogenesis and specialization [11].

Another perspective on geographical distribution can be obtained from analyzing culture collection deposits. The largest insect pathogen collection of the world is the USDA/ARS entomopathogenic fungi collection (ARSEF), which includes almost 15,000 occurrence records [12]. Although ARSEF has deposits from all over the world, most samples are from the USA, and then from Europe. We hypothesize that isolates from the rest of the world are less-represented due to lack of sampling. Despite the scarcity of well recorded data, at least 53 species out of the 125 valid EFPSZ species might be considered as cosmopolitan and at least 25 as ubiquitous (Figure 2, Table 1). Many of these might become ubiquitous as a result of worldwide spread of human agricultural activities, which spreads many crops worldwide along with their pests. One of the best examples of human mediated distribution might be P. gloeospora, found on several continents in mushroom growing farms [13].

3.2. Host specificity

EFPSZ fungi show a range of host-specificity. One third of EFPSZ parasitize two or more insect families. The species Erynia conica, E. rhizospora, E. selpulchralis, P. batallata, P. blunckii, P. echinospora, P. nourii, Z. aphidis, Z. canadensis are pathogenic to representatives of at least two families. An absolute generalist is Z. radicans, which infects insects in seven orders and 21 families (Table 1). However, two thirds of EFPSZ fungal species show some host-specificity and are able to infect only a narrower range of insects, usually attacking members of the same genus or family.

The flies (Order Diptera) are the most frequent hosts for EFPSZ fungi as more than one third of these fungal species were found killing Diptera. Nearly 25 percent of EFPSZ fungi infect insects in the order Hemiptera (30 pathogenic species), and nearly half that number were found infecting Coleoptera (16 pathogens) and Lepidoptera (15 pathogens). Within the Diptera, families most attacked by EFPSZ fungi are Calliphoridae (8 pathogen species), Tipulidae (7), Muscidae, Psychodidae and Sciaridae (6 each), and Chironomidae (5). In the genus Strongwellsea, species specialize exclusively on four dipteran families: Anthomyiidae, Muscidae, Sarcophagidae, and Scatophagidae. Among Hemiptera, the families Aphididae (14), Miridae (7), and Cicadellidae (6) are most infected by EFPSZ fungi. All other insect families have less than 5 pathogenic EFPSZ species infecting them (Figure 3).

3.3. Biological and ecological characteristics of EFPSZ fungi as biocenose components

The majority of species in the Erynioideae primarily infect insects in natural and agricultural environments. These habitats include aquatic biocenoses, forests and natural areas, and agrocenoses. It might be more precise to discuss the distribution of insect hosts, even if fungal infections can lead to infected insects relocating from their typical habitats [85]. Many EFPSZ species infect the imago (adult stage) of hosts. Among holometabolous hosts, species in the genus Strongwellsea only infect adults while some species from the other genera infect larvae. No EFPSZ species have been found attacking insect eggs.

These different host life cycle stages may occur in various ecosystems, so this factor should also be considered. In most cases, infected insects are found and collected on plant parts, partly due to the so-called climbing effect caused by any EFPSZ fungi [166]. This altered behavior may also be attributed to better visibility for researchers compared to the soil surface beneath vegetation. A comprehensive analysis of the distribution of entomophthoralean fungi in European biocenoses, focusing on forests and agrocenoses, was carried out by Bałazy [15]. Bałazy’s analysis emphasizes the importance of insect mobility, particularly because many insect hosts have wings and can migrate to neighboring ecosystems, spreading infection. This mobility is supported by collection of dispersing aphids infected with P. neoaphidis [167] and isolation of P. delphacis from planthoppers caught on a weather ship off the coast of Japan [75].

Most EFPSZ fungi are prevalent in aboveground ecosystems. The cadavers of insects infected with EFPSZ are often found on wild and cultured plants in various ecosystems. The presence of representatives of the genus Zoophthora in particular, is well-documented in numerous agricultural crops, orchards, and different types of forests. Species like P. dipterigena, P. phillonthii, Z. anglica, Z. miridis, Z. opomyzae, Z. petchii, Z. phytonomi, and Z. radicans are commonly observed in annual and perennial crops, meadows, pastures, orchards, and forests. These species are well-adapted to drier habitats. Species of the genera Strongwellsea and Zoophthora appear to be the most adaptable to a wide range of habitats, whether natural or human-created. Furthermore, aphid pathogenic species like P. neoaphidis and P. nouryi, have been found worldwide in many crops and are commonly observed at different temperatures and humidities.

All Entomophthorales require high humidity to release and disperse their conidia [168]. Interestingly, half of the species in the genus Erynia were found in aquatic or notably moist areas, e.g. E. aquatica, E. conica, E. curvispora, E. nematoceris, E. ovispora, E. sepulchralis, and E. variabilis. These species may serve as efficient biological control agents for insects requiring aquatic habitats during specific life stages due to their higher humidity needs compared to other species in this group. Additionally, five species in the genus Pandora, one species in Furia, and one species in Zoophthora were found in moist habitats. However, no Strongwellsea species were recorded in explicitly aquatic or moist environments (Table 1).

Soil is an unusual habitat for predominantly insect pathogenic EFPSZ. Nevertheless, at least one species, Pandora nouryi, infects root aphids (Pemphigus) and follows its hosts to this habitat, becoming a soil dweller [77]. Zoophthora myrmecophaga infects ants that move along their paths on the soil surface. Pandora brahminae, which infects scarabs inhabiting soil surface, also might be considered soil inhabitants.

3.4. Cultivability

Few species in the EFPSZ group have been isolated into pure culture, or even had their cultivability tested. Most species have been described only from insect cadavers and there are no cultures preserved. The largest culture collection of entomopathogenic fungi in the world is the USDA Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF, Ithaca, NY, USA), which contains over 14,000 fungal strains, isolated from infected insects or cadavers of insects dying from fungal infections. While most strains belong to the Ascomycota, entomophthoralean fungi are also well represented. This collection preserves 683 total isolates of EFPSZ [12]. These include 28 species known in the genera Erynia, Furia, Pandora, Strongwellsea and Zoophthora, as well as 36 isolates from these genera, which are not yet identified to the species level. Most species are represented by single or just a few isolates. However, there are over a hundred isolates representing species such as P. neoaphidis and Z. radicans, which reflects the easy cultivability of those species.

Few EFPSZ fungi can be cultivated on typical fungal nutritional media such as malt extract or potato dextrose agar or in the corresponding liquid media [168]. In the past, to ensure fungal growth of entomopathogens special media containing animal protein from additives such as liver, extracts of fresh or dried insects, blood serum, or egg yolk were used [21,169], providing the pathogens with specific nutrients absent in the usual laboratory media. Sometimes such rare and exotic media components as fly fat-bodies are used to stimulate spore germination or hyphal growth. Addition of yeast extract, arginine or other aminoacids to the medium substantially improves the growth of entomophthoralean fungi. Nowadays the most commonly used liquid medium for entomophthoralean growth is Grace′s Insect Medium (Sigma-Aldrich, St. Louis, USA), often with additives such as fetal bovine serum (ThermoFisher Scientific, Waltham, USA).

Larger scale production using simpler media has been developed for a few species. A method for producing Z. radicans dry-formulated mycelium has been developed with sporulation equivalent to cadavers. Recent examples of successful production on a large laboratory scale of the fungus P. cacopsyllae are the studies by Muskat et al. ([170,171], in press). This fungus that infects psyllids of the genus Cacopsylla has been fermented, encapsulated, and tested for above ground application.

The most fastidious EFPSZ species can so far only be cultured in vivo. They require the maintenance of an insect colony either in the laboratory or in the natural environment in order to maintain their population and complete their life cycle. The best results are obtained from using the natural hosts of the pathogenic fungus. In vivo production is the most difficult and labor intensive of methods for growing entomopathogenic fungi.

The ability to grow EFPSZ in culture is often connected with the ability of these fungi to infect insects at various stages of their life cycle, or at least stages other than the imago (adult), as seen in species like E. curvispora, Z. bialovienzenzis, Z. lanceolata, Z. phytonomi, and Z. radicans [15,21]. One of the remarkable characteristics of EFPSZ fungi, particularly those with high-host or life-stage specificity is the loss of their vigor and viability after several transfers on laboratory media despite strong initial growth [168,172]. These features of highly host-specific members of the EFPSZ can pose a challenge to mass production in biotechnology.

3.5. Genomics and Biotechnology

In addition to using these species directly for biological control, other potential biotechnology applications based on genes or proteins derived from these fungi are yet to be explored. The early diverging lineages of fungi, also defined as the paraphyletic group not including the Dikarya (e.g. Ascomycota and Basidiomycota), have emerged through the characterization of their genomes and identification of numerous shared genes and traits common to animals that were lost in more derived members of the Dikarya. Such homologs may in the future provide new insights into fundamental biology or even lead to therapeutics for human health. Fungi themselves are also known to produce diverse secondary metabolites/natural products, some of which are used commercially. Genome sequences have revealed evidence of greater biosynthetic capability for such molecules, even among some lineages of early diverging fungi [173]

At present, just a single species has had its genome sequenced from the Erynioideae, i.e. Z. radicans [174], due to the ease of its cultivability. The genome was generated as part of a large collection of species in a study that addressed the ploidy levels of the early fungal lineages, and so features about what this genome contains have not been described yet in detail. This genome sequence carries the information for how Z. radicans completes its lifecycle, including as an entomopathogen, but the mechanism is not immediately obvious. The Z. radicans genome is large, with the assembly at over 650 Mb currently divided over nearly 7,000 scaffolds and estimated to encode over 14,000 genes. The large size is due to the large amount of repetitive DNA within the genome (Figure 4). The entomopathogens in the Hypocreales (Ascomycota) contain numerous gene clusters for the synthesis of secondary metabolites, one of the best known being the cluster for the immunosuppressive cyclosporin from Tolypocladium inflatum [175], as well as other types of toxins (e.g. enterotoxins) with possible roles in altering host behavior [176]. However, such detailed information based on the Z. radicans genome is not yet available.

As pointed out above, one challenge toward generating more genome sequences is being able to obtain sufficient DNA, usually through culturing of isolates in vitro, which has not been possible for many of these species. Difficulties with culturing EFPSZ fungi make sequencing their genomes complicated because of the challenge of isolating high quality and high molecular weight DNA and RNA for sequencing. For many species, extraction of total DNA from the host insect cadaver might be the only option. However, the recent advances in single cell genomics [177], may provide a way in the near future to generate more genomic information about the genetic composition and potential virulence factors of EFPSZ species. Once identified, these genetic components could be utilized in vector-based expression systems for application as biopesticides. There are also a few genomes available for the closely related subfamily – Entomophthoroideae, including Entomophthora muscae, Entomophaga maimaga, Massospora cicadina as well as other species in Entomophthorales Conidiobolus coronatus Neoconidiobolus throboides and Basidiobolus meristosporus [178]. Summarizing all genome features for these Entomophthorales genomes, it can be predicted that the genomes of most EFPSZ fungi are much larger compared to the average ascomycete fungal genome size (40-60 kB) and can reach 600,000–1,000,000 kB in size and consist of many duplicated gene copies and repeated regions [179].

Figure 4. The Z. radicans genome features large amounts of repetitive DNA between coding regions. This figure is a modified version of the MycoCosm [180] visualization of 1 Mb of sequence on contig 1, showing in blue only 17 genes across the region, separated by repetitive DNA in black. Conservation of DNA sequences with two other sequenced species in the Entomophthorales is low.

Figure 4. The Z. radicans genome features large amounts of repetitive DNA between coding regions. This figure is a modified version of the MycoCosm [180] visualization of 1 Mb of sequence on contig 1, showing in blue only 17 genes across the region, separated by repetitive DNA in black. Conservation of DNA sequences with two other sequenced species in the Entomophthorales is low.

4. Discussion

The goal of using entomopathogenic fungi in various biotechnological applications, particularly to control populations of agricultural insect pests, has existed for several decades. However, entomophthoralean species have not been successfully developed and applied for biological control, despite numerous attempts. The major challenges in their application as biocontrol agents include the difficulty with cultivation of many species, requirements for specific abiotic conditions in the field, and the potentially low survival rates of these fungi outside of the host. However, EFPSZ fungi possess significant potential, which is still largely unexplored. Recent advances in genome sequencing technologies may allow researchers to access key genetic factors involved in virulence against insect hosts, in even those EFPSZ that cannot be easily cultivated, and biotechnology could then potentially be used to deploy these in various ways for pest control.

The ability of entomophthoralean species to infect insects from different families, or even from different orders, increases the diversity of target insect species for developing efficient biological control measures and selection of suitable pests to control. At the same time, the broad host-ranges of some of these fungi makes use of newly developed biological control remedies riskier as they may also affect other non-target insects, including those that are beneficial for natural ecosystems and humans.

While further research investigating host-range among EFPSZ is needed, the search for potential biocontrol agents within the Erynioideae might use information on current host-ranges and distributions, targeting hosts belonging to insect taxonomic groups that are known to be attacked by EFPSZ (Table 1). Even more important, prediction of possible non-targets of entomopathogenic species should account for insects within those same taxonomic groups in addition to pollinators and other beneficials. To some extent it is an advantage if a species already has been found on several continents and thus might be adapted for use in biological control over a large area. These species with high adaptability to various environments are promising candidates for targeting widespread insect hosts. Each genus of the EFPSZ group has several species that are distributed on at least three continents: in the genus Erynia, E. aquatica, E. conica, E. ovispora, and E. rhizospora. In the genus Furia, F. americana F. gastropachae, F. ithacensis, and F. virescens; and in the genus Pandora, P. blunckii, P. bullata, P. delphacis, P. dipterigena, P. gammae, P. neoaphidis, and P. nouryi. Many ubiquitous species are included in the genus Zoophthora: Z. aphids, Z. geometralis, Z. occidentalis, Z. phalloides, Z. phytonomi and Z. radicans. At least two species of the Strongwellsea, S. castrans and S. magna have been found on two continents. With increasing research on entomophthoralean fungi, we hypothesize that it is likely that a larger number of ubiquitous species will be identified.

Representatives of the genera Erynia, Pandora and Zoophthora are among important infective agents of insects under field conditions. The attempts at application of EFPSZ species on different continents has had success with Z. radicans in Australia using a strain originated from Israel to control the spotted alfalfa aphid, Therioaphis maculata [77].

A lower need for humidity could be considered an advantageous feature for potential biological control preparations using EFPSZ species. Aquatic species like E. aquatica might be successfully used only in wet habitats where they are highly adapted to the moist environment, and this could restrict application compared to the species found in diverse ecosystems. The broad ecological and geographical range of Z. radicans, recorded from numerous agricultural and natural habitats makes this species unique within the EFPSZ.

Cultivability is perhaps the main factor that determines the potential success of any biotechnological application with EFPSZ. If the fungus is hard to cultivate on artificial media, then the only way to apply it as a biological control agent is to keep it in vivo, infecting the insect population either in nature or under lab conditions. However, this is costly, cumbersome, and risky. Development of the biotechnological process and especially scaling it up demands easy culturing without losing virulence. The challenges of isolation into pure culture of the majority of EFPSZ species along with loss of vigor during numerous culture transfers significantly complicates the research on and development of potential biological control.

A problem with the successful pest control with many fungi is the level of susceptibility of the active organisms to external factors, such as fluctuations in temperature, humidity, and rainfall. Climate changes may significantly impact the relationship between fungi, insects and crops, and the interactions among them [181]. Furthermore, additional information needed for the eventual production of EFPSZ as biopesticides would be development of optimal methods for formulation and application.

5. Conclusions

Analysis of the available data on virulence, growth in vivo and in vitro, formulation, and field-testing suggests that one promising candidate for the development of an efficient biological control agents would be the species Z. radicans. This species seems to have potential for control of a range of lepidopteran larvae in many agricultural and forest ecosystems. Another species, P. neoaphidis, has great potential for control of numerous aphid species in cereals and legumes. Of special importance is the worldwide distribution of aphids impacting crops, and thus, the large market that exists. In orchards, P. cacopsyllae has recently proven to possess a potential for control of psyllid pests. A major objective here is that fruits are high value crops, that may favor biological control options. For the moist and aquatic habitats, there are several species infecting dipterans. Erynia aquatica and E. conica may have potential for mosquito control, however little is known about their virulence and growth in vitro. Obviously, the aforementioned factors are not the only factors determining the success or failure of biocontrol development; however, they play essential roles. However, advances in genome sequencing methods may allow researchers to access virulence factors and other genetic factors fro these fungi that could be harnessed for future biotechnological solutions.