Elsevier

Fungal Biology

Volume 124, Issue 5, May 2020, Pages 418-426
Fungal Biology

Serendipity in the wrestle between Trichoderma and Metarhizium

https://doi.org/10.1016/j.funbio.2020.01.002Get rights and content

Abstract

The fungal species Trichoderma is frequently found in soil antagonizing plant-pathogenic fungi as well as parasitizing plant-pathogenic nematodes. Metarhizium species are insect-pathogenic fungi that are used throughout the world to control agricultural insect pests. Here, we determine whether the antagonism (A) of Trichoderma atroviride to Metarhizium robertsii during growth and spore formation can impact the stress biology of M. robertsii conidia. Cultures of M. robertsii were either produced without exposure to T. atroviride (control) or in the presence of T. atroviride. M. robertsii was grown in dual culture with T. atroviride on potato dextrose agar (PDA) using the following treatments: 1) Trichoderma inoculated at the same time with Metarhizium (A0); 2) Trichoderma inoculated two days after the inoculation of Metarhizium (A2); 3) Trichoderma inoculated four days after Metarhizium (A4); 4) Trichoderma inoculated 6 d after Metarhizium (A6); 5) M. robertsii grown alone on PDA medium (control); and 6) M. robertsii grown alone on minimal medium (Czapek–Dox medium without sucrose) (MM). Germination of M. robertsii conidia from all six treatments was then assessed under osmotic, oxidative, UV-B, and thermal stress. M. robertsii conidia produced on MM were the most tolerant to all stress conditions. For all stress conditions, conidia from treatments A0 and A2 were not viable. For osmotic stress, conidia produced in treatment A4 were the most tolerant, followed by conidia from treatment A6, which were both more tolerant than the control. For oxidative stress, conidia produced in both A4 and A6 treatments were similarly tolerant and more tolerant than conidia produced in the control. For thermal stress, conidia produced in treatments A4, A6, and control (PDA) were similarly heat-tolerant. For UV-B stress, conidia produced in treatments A4 and A6 were equally tolerant and more tolerant than conidia produced in the control. The germination speed of conidia produced in all treatments, A0, A2, A4, and A6 was also tested. Conidia produced on MM germinated faster than the other treatments. Conidia produced in the A4 treatment were the second fastest, followed by conidia produced in treatment A6. Both A4 and A6 conidia germinated faster than conidia produced in the control treatment. Conidia produced in the treatments A0 and A2 did not germinate in 24 h. In summary, moderate levels of biotic stress from a fungal competitor or low-nutrient conditions can enhance the stress tolerance of M. robertsii conidia.

Introduction

Metarhizium robertsii is an important biological control agent and has been used worldwide to control insect pests for crop protection (Li et al., 2010, Rangel et al., 2015a, Rangel et al., 2018, Vega et al., 2009). These fungi need to be highly stress-tolerant to endure osmotic stress, oxidative stress, UV stress, heat stress, and other environmental challenges to be useful for control of insects in the agricultural environment (Araújo et al., 2018, Araújo et al., 2019, Azevedo et al., 2014, Dias et al., 2018, Souza et al., 2014).

Exposure of M. robertsii during mycelial growth to one type of stress can induce higher conidial tolerance to the same stress and other stress conditions – this phenomenon has been called cross-protection (Rangel, 2011, Rangel et al., 2018, Zhang et al., 2018). Conidia of M. robertsii are more tolerant to UV-B radiation or heat stress when they are produced under nutritive (Rangel et al., 2006), osmotic (Rangel et al., 2008b), alkaline (Rangel et al., 2015b), acid (Rangel et al., 2015b), and oxidative stresses (Rangel et al., 2008b) as well as illumination (Dias et al., 2019, Rangel et al., 2011), and hypoxia (Rangel et al., 2015b). Conidia of M. robertsii that are produced under stress are also more virulent to insects (Oliveira et al., 2018, Oliveira and Rangel, 2018, Rangel et al., 2008a). Hence, manipulation of the fungal physiology may improve conidial stress tolerance and virulence of insect pathogens, which are employed to control insect pests, increasing their ability to cope with the stressful agricultural and forestal environments.

Trichoderma species often dominate communities of saprotrophs and many other fungal niches found in soils (Chen and Zhuang, 2017, Druzhinina et al., 2011). They also exhibit the characteristics of a zymogenous species. The word zymogenous was coined by Sergei Winogradsky (Winogradsky, 1924, Winogradsky, 1949) for those microorganisms that exhibit high levels of activity and rapid growth on easy substrates that become available in the environment (Atlas and Bartha, 1998). This necrotrophic and mycoparasitic fungus is known to colonize the soil (Harman, 2000), produce substances that act as plant-growth promoters (Druzhinina et al., 2011, Martínez-Medina et al., 2014), occur as endophytic symbionts of plants (Busby et al., 2016), act as insect pathogens (Ghosh and Pal, 2016), kill plant-parasitic nematodes, inhabit diverse environments including wood, bark, and mushrooms (Druzhinina et al., 2011), and is commonly found in flooded houses (Bennett, 2015).

Competition between fungi may occur remotely and be mediated by volatile organic compounds (VOCs, including aliphatic alcohols, aldehydes, ketones, pyrones, and aromatic compounds) and secondary metabolites (Bennett and Inamdar, 2015, Druzhinina et al., 2011, Fox and Howlett, 2008, Hung et al., 2015, Knowles et al., 2019). In addition, many attack and defense mechanisms recruited by mycelia interacting under fungal co-culturing are found, which include competition for space and nutrients; direct parasitism; protein stabilization and recycling; production of reactive oxygen species (ROS); production of inhibitory biosurfactants; detoxification of toxic metabolites; and sealing of mycelial front (Druzhinina et al., 2011, Ujor et al., 2018). In addition, microbes can be inhibited by cell wall-degrading enzymes produced by Trichoderma spp, such as chitinase, β-1,3 glucanases, pectinase, lipase, amylase, α-1,2-mannosidase, protease, and N-Acetyl-β-d-glucosaminidase (Saxena et al., 2015, Troian et al., 2014).

Both Metarhizium and Trichoderma may sense each other by quorum sensing. Quorum sensing is a mechanism of microbial communication in which accumulation of signaling molecules enables a cell to sense a cell density (Padder et al., 2018). Quorum sensing is mediated by small diffusible signaling molecules that accumulate in the extracellular environment (Hogan, 2006). The first fungi reported to have a quorum-sensing system is Candida albicans, which produce a molecule identified as farnesol, a compound that blocks the yeast-to-mycelia transition (Hornby et al., 2001). Farnesol was found to have detrimental effects on many bacteria and other fungi (Albuquerque and Casadevall, 2012, Padder et al., 2018), including in Metarhizium rileyi at high concentrations (>2 mM farnesol) (Boucias et al., 2016). Nevertheless, nothing is known about quorum sensing between interspecific mycelial interactions of Trichoderma and Metarhizium.

Considerable research has been conducted on the competitive interactions between fungi (Druzhinina et al., 2011). However, nothing is known about whether growth under biotic stress, by interspecific mycelial interactions, can induce cross-protection against abiotic stresses. The current study focused on the implications of biotic stress in M. robertsii caused by Trichoderma atroviride and analyzed the tolerance of M. robertsii conidia to osmotic, oxidative, UV-, and high-temperature stresses after the fungus was exposed to biotic stress (induced by competition with T. atroviride). The specific objectives were to determine: 1) Does biotic stress from T. atroviride influence the germination speed of M. robertsii conidia? and 2) Is Metarhizium conidia produced in dual culture with Trichoderma more tolerant to different stress conditions?

Section snippets

Fungal isolates

M. robertsii (ARSEF 2575) was obtained from the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF; Robert W. Holley Center for Agriculture & Health, Ithaca, NY, USA) and T. atroviride (IMI, 206040) was generously provided by Alfredo H. Herrera-Estrella (Laboratorio Nacional de Genómica para la Biodiversidad, Mexico) (Casas-Flores et al., 2006). This strain was isolated from Picea abies (Norway spruce) in Sweden in 1970 and has been registered and used in Europe as a biopesticide (

Results

The nutritive stress caused by the minimal medium produced conidia that were more tolerant to all stress conditions than all other treatment with biotic stress caused by Trichoderma (Fig. 2, Fig. 3, Fig. 4, Fig. 5). For all stress conditions, conidia from treatments A0 and A2 were not viable.

For osmotic stress, M. robertsii conidia inoculated four days after T. atroviride (A4) were the most tolerant, followed by conidia inoculated six days after the antagonist treatment (A6). Conidia of

Discussion

Metarhizium and Trichoderma species frequently occur together in natural environments such as the rhizosphere (Hu and St. Leger, 2002, Liao et al., 2013, Razinger et al., 2014, Wiberth et al., 2019). Trichoderma species can completely colonize plant-pathogens such as Sclerotium rolfsii (Hirpara et al., 2016) and Fusarium oxysporum (Martínez-Medina et al., 2014). However, T. atroviride was not able to replace the fungus M. robertsii in dual culture even at early dual-inoculations of Trichoderma

Acknowledgments

We are grateful to Dr. Richard A. Humber (USDA-ARS Collection of Entomopathogenic Fungal Cultures, R.W. Holley Center for Agriculture & Health, Ithaca, NY) for the fungal isolate ARSEF 2575 of Metarhizium robertsii. The mycoparasitic and biotrophic fungal species isolate IMI 20604 of Trichoderma atroviride was provided by Dr. Alfredo H. Herrera-Estrella, Laboratorio Nacional de Genómica para la Biodiversidad (CINVESTAV). The authors also thank Alene Alder-Rangel at Alder’s English Services for

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