Abstract Environmental stresses can compromise the interactions of plants with beneficial microbes. In the present review, experimental results showing that stresses negatively affect the abundance and/or functionality of plant beneficial microbes are summarized. It is proposed that the environmental interference of these plant−microbe interactions is explained by the stress‐mediated induction of plant signalling pathways associated with defence hormones and reactive oxygen species. These plant responses are recognized to regulate beneficial microbes within plants. The direct negative effect of stresses on microbes may also contribute to the environmental regulation of these plant mutualisms. It is also posited that, in stress situations, beneficial microbes harbour mechanisms that contribute to maintain the mutualistic associations. Beneficial microbes produce effector proteins and increase the antioxidant levels in plants that counteract the detrimental effects of plant stress responses on them. In addition, they deliver specific stress‐protective mechanisms that assist to their plant hosts to mitigate the negative effects of stresses. Our study contributes to understanding how environmental stresses affect plant−microbe interactions and highlights why beneficial microbes can still deliver benefits to plants in stressful environments.

Environmental interference of plant−microbe interactions

Abstract
 Plants provide a unique ecological niche for diverse communities of fungal endophytes that vary in their impact, positive to negative, on the host plant. Fungal endophytes colonize plants without any visible disease symptoms for at least part of their life history. These symbionts are critical components of natural and semi-natural ecosystems, as well as cultivated agricultural communities, dramatically influencing plant adaptation and evolution. Many temperate grass species are reliant on their mutualistic association with obligate fungal endophytes of the genus Epichloë for a variety of fitness benefits, such as persistence under both invertebrate and vertebrate grazing pressure and improved adaptation to abiotic stresses. In New Zealand, Epichloë endophytes have been estimated to contribute $200 m per year in increased animal production through improved pasture persistence and yield. Other fungal endophytes have also been studied in temperate grasses, although much less is understood about the biological and economic impacts of these plant-fungal interactions. Determining which fungal endophytes are mutualistic has been, and continues to be, a challenge, particularly as we begin to realize that endophytes interact with other endophytes and little is understood about what factors shape endophyte community structures.

Improved adaptation of temperate grasses through mutualism with fungal endophytes.

Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand’s agricultural economy by conferring biotic advantages to the host grass. Overall, Epichloë endophytes contribute NZ$200 million to the economy annually, with strain AR37 estimated to contribute NZ$3.6 billion to the New Zealand economy over a 20-year period. This strain produces secondary metabolites, including epoxyjanthitrems, which are a class of indole diterpenes, associated with the observed effects of AR37 on livestock and insect pests. Until very recently, AR37 was intractable to genetic modification but this has changed with the application of CRISPR-Cas9 based gene editing techniques. In this paper, gene inactivation by CRISPR-Cas9 was used to deconvolute the genetic basis for epoxyjanthitrem biosynthesis, including creating an AR37 strain that has been edited to remove the biosynthesis of all indole diterpenes. We show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) through an AMA1-based plasmid that simultaneously expresses the CRISPR-Cas9 system and selectable marker. Genetic modification events in these transformants were investigated through genome sequencing and in planta chemistry.

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Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing

Asexual Epichloe are obligate fungal mutualists that form symbiosis with many temperate grass species, providing several advantages to the host. These advantages include protection against vertebrate and invertebrate herbivores (i.e., grazing livestock and invertebrate pests, respectively), improved resistance to phytopathogens, increased adaptation to drought stress, nutrient deficiency, and heavy metal-containing soils. Selected Epichloe strains are utilised in agriculture mainly for their pest resistance traits, which are moderated via the production of Epichloe-derived secondary metabolites. For pastoral agriculture, the use of these endophyte infected grasses requires the balancing of protection against insect pests with reduced impacts on animal health and welfare.

Asexual Epichloë Fungi—Obligate Mutualists

Asexual Epichloë are endophytic fungi that form mutualistic symbioses with cool-season grasses, conferring to their hosts protection against biotic and abiotic stresses. Symbioses are maintained between grass generations as hyphae are vertically transmitted from parent to progeny plants through seed. However, endophyte transmission to the seed is an imperfect process where not all seeds become infected. The mechanisms underpinning the varying efficiencies of seed transmission are poorly understood. Host gene expression in response to Epichloë sp. LpTG-3 strain AR37 was examined within inflorescence primordia and ovaries of high and low endophyte transmission genotypes within a single population of perennial ryegrass. A genome-wide association study was conducted to identify population-level single nucleotide polymorphisms (SNPs) and associated genes correlated with vertical transmission efficiency. For low transmitters of AR37, upregulation of perennial ryegrass receptor-like kinases and resistance genes, typically associated with phytopathogen detection, comprised the largest group of differentially expressed genes (DEGs) in both inflorescence primordia and ovaries. DEGs involved in signaling and plant defense responses, such as cell wall modification, secondary metabolism, and reactive oxygen activities were also abundant. Transmission-associated SNPs were associated with genes for which gene ontology analysis identified “response to fungus” as the most significantly enriched term. Moreover, endophyte biomass as measured by quantitative PCR of Epichloë non-ribosomal peptide synthetase genes, was significantly lower in reproductive tissues of low-transmission hosts compared to high-transmission hosts. Endophyte seed-transmission efficiency appears to be influenced primarily by plant defense responses which reduce endophyte colonization of host reproductive tissues.

https://www.frontiersin.org/articles/10.3389/fpls.2022.1025698/pdf

Epichloë seed transmission efficiency is influenced by plant defense response mechanisms

Epichloe–grass associations are among the most well understood aboveground plant-microbe symbioses. Current attention has focused on understanding the molecular signals that underpin how these endophytes interact with their grass hosts to form natural and lifelong mutualistic symbioses. We review features of these symbioses, focusing on understanding host and endophyte responses, and their interactions which ultimately deliver important fitness benefits. One of the main selective forces of this symbiotic relationship are the endophyte expressed secondary metabolites. We examine how the host influences expression of these compounds and explore their function. Understanding the complex regulation of these symbioses at the cellular, biochemical, and molecular level is advancing and we show how such responses and subsequent outcomes are influenced by the evolutionary relationships between endophytes and hosts. We reveal a dynamic symbiosis that undergoes significant host reprogramming to accommodate its protective symbiont throughout the different stages of the host plant life cycle.

The dynamic mechanisms underpinning symbiotic Epichloë–grass interactions: implications for sustainable and resilient agriculture

The wheat sheath miner, Cerodontha australis Malloch (Diptera: Agromyzidae), is a small leaf‐ and stem‐mining fly that is native to New Zealand and eastern Australia where it feeds on grasses and cereals. Although not considered to be a major insect pest of pastures in New Zealand, it can be abundant during spring and summer in Waikato, New Zealand, and infests tillers of the introduced pasture plant, perennial ryegrass, Lolium perenne L. (Poaceae). Cerodontha australis is not deterred from feeding on L. perenne infected with commercially available strains of the fungal endophyte Epichloë festucae var. lolii, which have been shown to provide protection from a range of other insect pests. In this study, three‐pot trials and periodic examination over 18 months of field‐collected L. perenne tillers showed that two Epichloë endophyte strains, AR47 and AR48, reduced the survival and feeding damage by C. australis larvae, but there was no evidence of an effect on adult female feeding or oviposition. Compared to other strains of E. festucae var. lolii endophytes tested, plants infected with AR47 and AR48 had fewer and smaller larvae and less larval mining. The number of tillers with mining was reduced in AR47 relative to endophyte‐free (Nil) by 57.1% (ranging from 25.0 to 92.5% across trials) and in AR48 by 67.1% (26.3–100%). Larvae in AR47 and AR48 were found further from the base of the tiller where they were less likely to damage the meristem and cause tiller death. The number of larvae and pupae in pseudostems was reduced in AR47 and AR48 compared with Nil by 78.4 and 82.0%, respectively, in the field trial. Very few larvae survived to pupation in AR47 and AR48 plants, with a reduction compared to Nil of over 81.5% in AR47 and over 97% in AR48 in each of the four trials presented here.

Taryn A. Miller

Papers

The dynamic mechanisms underpinning symbiotic Epichloë–grass interactions: implications for sustainable and resilient agriculture

Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing

Two Epichloë festucae var. lolii endophytes in Lolium perenne reduce larval populations of the wheat sheath miner, Cerodontha australis