Biological Pesticide-free Pest And Pathogen Control

Abstract

Ants have been proposed as a sustainable alternative to pesticides in conventional agriculture, as they supposedly possess some intrinsic defense mechanisms against infections, that might be utilized in protecting farmed plants against pests and pathogens. Previous studies on ant-associated plants have shown an increase in herbivore damage and pathogen infection following exclusion of some naturally occurring ants. However, the actual mechanisms of protection against pathogens remain unknown, and thus the utility of adding ants to plants not usually associated with ants, such as many conventional crops, is unclear.

In this study, we transplanted two species of weaver ants (genus: Oecophylla) onto a non-ant-associated plant, Arabidopsis thaliana, either prior to or after infection with a plant-parasitic oomycete, Albugo sp. We hypothesized that the ants would negatively influence the extent of pathogen infection. Results showed that the plants that were treated with ants experienced a non-significant decrease in percentage leaf area infected by the pathogen compared to the plants that had not been treated with ants. This applied regardless of if the ant treatment was applied before or after infection of the plants. In conclusion, our study, in addition to previous studies on the topic, suffers the drawbacks from lack of knowledge on the general mechanisms and behavioral aspects of ants interacting with plants and plant pathogens. Further studies are needed if ants are to be successfully implemented in sustainable biological pathogen management in agriculture.

Introduction

The human population recently reached 7 billion and the number continues to rise (Roser & Ortiz-Ospina, 2018). The recent explosive population growth creates a need for more efficient, high-yielding agriculture to feed every mouth, but it has also caused increased environmental pressure. Conventional agricultural methods include the use of chemical fertilizers and pesticides to increase crop yields. However, the pesticides used to control growth of undesired plants and avoid pests and disease, also harm the surrounding environment: Pollution of soil, water and air, as well as general loss of biodiversity are all known side effects of pesticide use (van der Sluijs et al., 2015), and several health problems in humans have been linked to pesticide exposure as well (Gilden, Huffling, & Sattler, 2010).

Agriculture with reduced pesticide use is usually characterized by lower yields and higher costs (Seufert, Ramankutty, & Foley, 2012). Weeds are eliminated mainly through cultivation of the soil or manual weeding, and pests and diseases are sought to be prevented through crop rotation and mixed cropping, as well as limited chemical control by use of a few naturally occurring substances (Migliorini & Wezel, 2017). These processes are more labor-intensive, which makes agriculture with reduced pesticide use less effective than the conventional practices. Consequently, development of sustainable agricultural methods of equal or greater effectivity than conventional methods are in high demand.

One method of biological pesticide-free pest and pathogen control has been suggested by Offenberg (2015): Ants. Ants are social insects and therefore they are, as all other organisms living in groups, particularly vulnerable to pathogen transmission, due to a high number of interactions between closely related individuals in the group. This threat of disease creates an extraordinary selection pressure and has resulted in an array of behavioral and chemical defence mechanisms in ants, as reviewed in Tranter and Hughes (2015).

The defenses range from self-grooming, to remove parasites e.g., to antimicrobial glandular secretions or acidic venom, which the ants apply to themselves (Tranter & Hughes, 2015) or spread around in their nests and on nesting materials (Worsley et al., 2018) to sterilize them. And, from a small number of recent studies on myrmecophytes - plants that live in mutualistic associations with ant colonies - it appears that ants not only defend themselves and their colony against diseases, but possibly also protect their near vicinity, such as their host plant, against pests and pathogens. Gonzalez-Teuber, Kaltenpoth, and Boland (2014) showed a correlation between presence of ants (Pseudomyrmex) and lowered levels of pathogen-inflicted leaf damage on the host plant, and Heil, Baumann, Andary, Linsenmair, and McKey (2002) likewise found a correlation between presence of ants (Crematogaster) and the inability to infect the ant host plant with fungi. Offenberg (2015) also scrutinize several studies in which weaver ants (Oecophylla) have been found to have an effect on pests and pest damage in several types of plants and trees. These previous studies have been performed on species of plants naturally inhabited by ants: Either the naturally occurring ants were excluded from some plants to act as controls, or plants which - by chance - were not inhabited by ants were compared with plants of the same species that were.

The focus of these studies has mainly been on the effect of ants on selected insect pests or pathogens and not on the actual mechanism by which the ants affect said pests and pathogens. To decide on the versatility of ants as sustainable alternative to pesticides, however, it is crucial to determine the underlying mechanisms of these apparent pesticide-like effects. Fundamentally, the ants can either provide protection actively or passively. Considering the concept of mutualism, it can be described as a mutually beneficial interaction between two species (Bronstein, 1998). Thus, it is entirely conceivable that the ants will only protect their host plants when the plants provide something in return, such as nectar or housing structures. In such case, you would expect the anti-pathogen effect of the ants to originate from an active effort; the ants might eat the pest or the pathogen spores, or they might secrete anti-bacterial substances onto infected plant material. Anti-pathogen effects generated in this way might prove hard to utilize in agriculture, as many farmed plants are not myrmecophytic and do not possess e.g. extrafloral nectaries to attract and reward ants.

Yet, it is also possible that effects are prompted by the mere presence of ants, unconditional of their actual activities. Support for this hypothesis has been provided by Gonzalez-Teuber et al. (2014), who found an inhibitory effect on a number of bacteria, similar to that from exposure to living ants (Pseudomyrmex), from a methanol extract of ant legs. If the mechanisms of protection are indeed passive, the basis for implementation of ants in agriculture is much improved, as the ants would be expected to provide the same protection to all plants regardless of rewarding.

In this study, we aimed to explore the effect of the possible anti-pathogen effects of ants on non-myrmecophytic plants. We hypothesized that ants would negatively influence the extent of plant pathogen infections. Through transplantation of weaver ants onto Arabidopsis thaliana plants, we sought to evaluate the effect of ants on the extent of pathogen infection, when the ants were given access to plants either (a) after the plants were inoculated with a plant pathogen and disease had developed or (b) before the plants were inoculated with a plant pathogen.

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Methods and materials

The study system

Our study system consisted of Australian weaver ants (Oecophylla smaragdina) and African weaver ants (O. longinoda), a plant (Arabidopsis thaliana) and a plant-parasitic oomycete (Albugo sp.).

The weaver ants were chosen on the basis of availability and the fact that they are extremely well-studied (Offenberg, 2015). The arboreal weaver ants are easy to manage in a laboratory, as each colony can be contained on a single host plant surrounded by water. The ants are very territorial and will patrol and guard the outer bounds of their territory. This behavioral trait renders possible our method of creating our tests plants to the host plants, as the ants will incorporate the test plants into the territory and begin patrolling it, when they detect their presence.

The weaver ants used in the study were collected from Australia and Africa, respectively, by Joachim Offenberg and are currently kept on coffee plants (Coffea arabica) in a greenhouse at section for Plant and Insect Ecology, Department of Bioscience, Aarhus University located in Silkeborg, Denmark.

Our study plant is likewise a model organism of choice: A. thaliana is favored in plant biology research due to small size, short generation time and ability to self-pollinate, which makes it more-than-manageable in laboratory settings and easy to cultivate in large amounts (Koornneef & Meinke, 2010). Albugo sp. is a natural pathogen of plants in the Brassicaeceae, which includes A. thaliana (Choi, Shin, & Thines, 2009). As a pathogen Albugo causes a disease commonly known as white rust and produces chlorotic (white/yellow-ish) lesions.

Cultivation of plants

Two-week old seedlings were transplanted into plastic containers filled with substrate (Pindstrup Såjord); two seedlings per container, 94 seedlings total. The substrate of each container was thoroughly soaked in water prior to transplantation, and the plants were subsequently watered with 2-3-day intervals. The plants were distributed among two boxes and kept under a transparent lid to improve the humidity. The plants were kept in a light cabinet at section for Genetics, Ecology and Evolution, Department of Bioscience, Aarhus University - Aarhus, Denmark. The light cabinet provided 150 mmol·m^(-2)·s^(-1) of light and kept a temperature regime of 18 °C for 16 hours and 14 °C for the remaining 8 hours a day.

After 10 days of growth, the 88 plants of highest quality were moved into separate plastic pots.

Again, the substrate of the plant containers was thoroughly soaked in water, and plants watered with 2-3-day intervals and kept in boxes under a transparent lid. Additionally, we removed any emerging flowers from the plants - and kept on during so for the rest of the study period - as to prevent the plants from relocating their energy from growth towards flowering. From here on, the plants were kept in a light cabinet at section for Aquatic Biology, Department of Bioscience, Aarhus University - Aarhus, Denmark. The light cabinet provided 200 mmol·m^(-2)·s^(-1) of light and kept a temperature regime of 20 °C for 16 hours and 18 °C for the remaining 8 hours a day.

Inoculation of plants

We made spore suspensions from older plants, already heavily infested with Albugo sp. Large leaves containing many white blisters were carefully removed with tweezers and transferred to 15 ml. test tubes; six to seven leaves per tube, two tubes per sub-study. 5 ml. milli-Q water was added to each tube before shaking them thoroughly to submerge every leaf. The tubes were placed on ice for 90 minutes, causing the sporangia to burst and release spores into the water. The tube contents were then filtered through Miracloth to remove plant material.

The resulting spore suspensions were sprayed onto our study plants by airbrush; each plant was sprayed just shy of the dripping point. The plants were then placed into boxes (test plants and control plants allocated randomly) with transparent lids, and the boxes additionally covered in black plastic. The boxes were placed in a cold room at 4 °C for around 16 hours before being uncovered and relocated to the light cabinet at section for Aquatic Biology.