Endophytic fungi are microscopic fungi that live all or part of their lives inside plant tissues, both in cells and in the intercellular spaces of various plant parts, without causing damage to the host plant (Stone et al., 2004; Novotný, 2006). Endophytic fungi have a mutualistic relationship with plants, which means that both parties gain some benefit from this interaction (Saikkonen et al., 2010). For example, fungi can provide plants with substances that increase resistance to stress, such as drought, disease or pests. They can also produce substances that help plants survive in harsh conditions (Zhao et al., 2011). This may include the production of chemical compounds that repel herbivores or inhibit the growth of pathogens (Deshmukh et al., 2018). These chemicals can be various substances, such as alkaloids, terpenes, or antibiotics (Hashem et al., 2023; Sonowal et al., 2024). Endophytic fungi are found in various types of plants, from grasses to trees. This symbiotic interaction may be a key factor for plant survival in different environments (Yan et al., 2019).
Research on endophytic fungi is gaining importance in the field of agriculture and ecology (Baron & Rigobelo, 2022). Researchers are investigating how endophytic fungi can contribute to increased yields, improve plant resistance to pests and diseases, and promote sustainable agriculture (Liu-Xu et al., 2022). Overall, endophytic fungi represent an interesting and important aspect of plant biology and ecology, and their study can provide useful insights for agricultural improvement and plant protection.
Several species of fungi specialize in the endophytic way of life. One example is the endophytic fungi of the genus Epichloë, which are known for their symbiotic relationship with grasses (Saikkonen et al., 2016). Epichloë mushrooms can produce substances that are repellent or toxic to insects. This increases the plant's resistance to herbivores and can improve its survival (Laihonen et al., 2022). This symbiosis thus gives the plant an advantage in the fight against predators and promotes its growth. Endophytic fungi of the genus Epichloë can also affect the soil in which they grow (Pérez et al., 2020. Some species of these fungi can produce substances that affect the microbial community of the soil and can have an impact on the surrounding ecosystem (Mosaddeghi et al., 2021). Research has shown that there is genetic variability among different strains of Epichloë. This variability may influence the benefits of symbiosis and interactions with host plants (Realini et al., 2024). In the broader context of agricultural production, the symbiotic relationship with endophytic fungi of the genus Epichloë may be used to protect crops from pests. This can reduce the need to use chemical pesticides and increase crop resistance to stress conditions. Broadly speaking, endophytic fungi play a key role in ecosystems where they can influence the growth and health of host plants.
Alkaloids from the indole-diterpene group, produced by endophytic fungi of the genus Epichloë (Ludlow et al., 2019), are a large and diverse group of bioactive secondary metabolites that perform important ecological functions as defense and signaling molecules (Galindo-Solís et al., 2022). One of the most toxic indole diterpenes is a substance called lolitrem B (Philippe, 2016). Lolitrem B has the properties of a tremorogenic poison. Tremorogenic effects refer to the ability of certain substances to cause tremors, which are uncontrolled, rhythmic movements of a part of the body. Tremors can affect different parts of the body, including the hands, feet, head, or the whole body. There are several substances known for their ability to induce tremor, and one such substance is lolitrem B. After intraperitoneal administration of lolitrem B to experimental animals, a long-lasting tremor occurs that is very severe (Gallagher et al., 1998; Munday-Finch et al., 1999). Skeletal muscle electromyographic activity recorded in sheep treated with flitter B at 25 to 110 pg kg −1 had no effect at low doses, but at higher doses produced tremors that appeared within 20 to 30 minutes and persisted for more than 24 hours (Smith et al., 1997).
Mechanism of action of Lolitrem B
Pastures associated with toxic outbreaks contain several potentially neuroactive metabolites of the Lolitrem biosynthetic pathway and currently their role in the presentation, severity and duration of the clinical signs, their location, and mode of action in the brain has not been well defined (Munday-Finch et al., 1998; Reddy et al., 2019). The mechanism of action of Lolitrem B involves its interaction with large conductance calcium-activated potassium channels, specifically targeting the α subunit of these channels.
Lolitrem B targets the large conductance calcium-activated potassium channels (BK channels) and specifically interacts with the α subunit of these channels. BK channels play a crucial role in allowing neurons and other electrically sensitive cells to "reset" after firing. By blocking these channels, Lolitrem B prevents neurons or heart cells from firing again, affecting nerve and heart function. Inhibition of BK channels by Lolitrem B can lead to increased release of excitatory neurotransmitters, resulting in symptoms such as ataxia, hypersensitivity, increased smooth muscle contraction in the colon, and an increased heart rate. The compound's action on these channels can explain the observed effects on nerve and heart function in animals exposed to Lolitrem B. Lolitrem B is not very soluble and tends to be stored in fat after ingestion. This slow release from fat contributes to the gradual onset and lingering effects of Lolitrem B toxicity. The more Lolitrem B is ingested, the more is stored in fat, leading to a higher concentration of the compound in the body. Studies have shown that Lolitrem B accumulates specifically in certain brain regions like the cerebral cortex and thalamus. The compound concentrates in these regions over time, with clearance potentially occurring over a longer period than analyzed in studies. This brain accumulation contributes to the neurological effects observed in animals exposed to Lolitrem B (Reddy et al., 2019).
In summary, Lolitrem B exerts its toxic effects by blocking BK channels, disrupting normal nerve and heart function by interfering with neurotransmitter release. The compound's bioaccumulation in specific brain regions further contributes to its neurotoxic effects observed in animals affected by perennial ryegrass staggers.
Symptoms of Lolitrem Poisoning
The symptoms of Lolitrem B poisoning in animals, particularly livestock like sheep, cattle, and horses, can vary depending on the dosage and severity of exposure. The symptoms of Lolitrem B poisoning can take some time to appear, as the compound's effects come on slowly after ingestion. Lolitrem B poisoning can result in a range of symptoms in livestock, from mild tremors to severe staggering and collapse, with potential fatal outcomes at high concentrations. Understanding these symptoms is crucial for early detection and intervention to mitigate the health risks associated with exposure to this neurotoxic compound (Reddy et al., 2019).
Treatment for Lolitrem B
As of now, there is no established antidote for Lolitrem B poisoning. In cases of exposure to this tremorgenic agent, the primary and practical treatment revolves around removing affected animals from the toxic pasture and substituting their diet with non-contaminated feed. This approach aims to prevent further ingestion of Lolitrem B and mitigate the impact of its toxic effects on the animals. Given the absence of a specific antidote, prompt action in relocating animals to a safe environment and providing alternative nourishment stands as the most effective and feasible course of intervention (Massey University, 2008).
The toxicology assessment conducted by Finch et al. (2022) marked a significant milestone by revealing that dose rates of Lolitrem B and the epoxyjanthitrem-producing endophyte, AR37, administered to mice were substantially higher than those realistically achievable through the consumption of animal products by humans. Importantly, no toxicity was observed in mice at these elevated dose rates. This finding, coupled with the concentrations reported in animal products, led the study to conclude that neither Lolitrem B nor the AR37 endophyte raised any discernible concerns for human health. Nevertheless, it is crucial to acknowledge the potential for species differences in toxin metabolism.
As new endophytes are consistently entering commercial use, each endophyte-grass association generates a unique profile of secondary metabolites. While the current evaluation ensures the safety of these novel endophytes in animals, a comprehensive assessment of food safety and potential implications for human health remains imperative. The evolving landscape of endophyte research underscores the necessity for ongoing scrutiny and consideration in the context of broader public health concerns (Finch et al., 2022).
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