Previous studies revealed that endophytes improve host tolerance to stress through a variety of morphological and physiological adaptations and adjustments by promoting host growth and photosynthesis, increasing the levels of beneficial metabolites, activating antioxidant systems to scavenge ROS, modulating plant growth phytohormones, improving nutrient uptake, and maintaining ionic homeostasis (Malinowski et al. 2000, 2019; Song et al. 2015; Hume et al. 2016; Nagabhyru et al. 2013). In the present study, we evaluated the plant growth, contents of phytohormones, stoichiometry of C, N and P, and Na+ and K+ ion transport in perennial ryegrass under different treatments. The results showed that endophyte only regulated phytohormones, the rational distribution of ion content and nutrient elements in host plants of both mutation and wild types under some treatments; however, its role was inconsistent depend on treatments. Endophytes had either beneficial or harmful effects on plant stress tolerance under certain treatments.
The effects of stress and endophyte on the growth of perennial ryegrass
In the present study, stress treatments inhibited the growth of perennial ryegrass. While, E. festucae var. lolii only had beneficial effects on host growth for wild type plants, but had no effects on mutant plants which suggested the mutation treatment may have effects on endophyte function. Some previous studies have indicated that the effects of endophytes on the growth of perennial ryegrass may be either beneficial or harmful, depending on a combination of biotic and abiotic factors, as well as the interaction between the host and endophyte genotypes (Marks et al. 1991; Cheplick 1997, 1998).
The effects of stress and endophyte on hormones contents of perennial ryegrass
In the present study, both stress treatments and endophytes had no significant effect on IAA and ABA contents, however, had significant effects on GA and SA contents. These changes in endogenous hormones in plants under different conditions confirmed that plants utilise hormones in response to stress. Phytohormones are important signalling molecules that are related to plant growth and physiological and developmental processes (Aaron et al. 2009). IAA is the most common auxin produced by plants, and its concentration is key in the regulation of plant growth and development (Müller 2003). ABA is known to induce stomatal closure as a water-conserving response, in which plants benefit in the short term by reducing water loss via transpiration (Lemichez et al. 2001). CTK plays a key role in improving grain yield by affecting the source/sink transition (Peleg et al. 2011). GA is a vital plant growth regulator, which plays an important role in seed germination growth of floral organs, and in lateral shoot formation, which is also observed to encourage plant growth and improvement under numerous abiotic stress conditions (Olszewski et al. 2002; Tuna et al. 2008; Ahmad et al. 2010). SA is a well-known signalling molecule that affects plant growth and development and is involved in plant immune system responses to pathogens and insect herbivores (Hayat et al. 2010; Bastías et al. 2018).
In the present study, Epichloë endophyte different effects on CTK, GA and SA contents; this is consistent with previous studies, which revealed that Epichloë endophytes change hormones to improve host stress tolerance (De Battista et al. 1990; Saikkonen et al. 2004, 2013; Xia et al. 2018). Bunyard and McInnis (1990) reported that E+ tall fescue plants produced significantly more ABA in response to drought stress than E- plants. Some glasshouse-based studies have indicated a similar endophyte-enhancement of ABA concentration in tall fescue leaf tissue in response to drought (Joost 1995). Similar results were also observed in some Chinese native grasses, such as Achnatherum inebrians and Festuca sinensis. E+ Chinese wildrye (Leymus chinensis) plants have a higher SA content than E- plants, especially when they are exposed to B. sorokiniana and C. lunata (Wang et al. 2016). Some endophytes have been reported to produce IAA and related indole compounds in cultures (De Battista et al. 1990, Yue et al. 2000). Phytohormone production in planta may also induce defense-related secondary metabolism in plants. However, very little is known about the role of hormones in symbiosis and their direct effects on host fitness traits.
The effects of stress and endophyte on the Na+/K+ transport of perennial ryegrass
In the present study, compared with the control, Na+ ion content significantly increased (P< 0.05), whereas K+ ion content significantly decreased (P< 0.05) under stress treatments. Na+/ K+ and SK, Na also significantly increased (P< 0.05) under stress treatments. Ca2+ levels did not change in most plants. Accumulation of inorganic ions, such as Na+ and K+, is one of the mechanisms for osmotic adjustment in plants during stress response (Shabala et al. 2011). This is very important for alleviating host damage because Na+ accumulation in plant cells causes extreme damages by inhibiting enzymes, disrupting K+ acquisition, inhibiting K+-dependent metabolic processes, and causing oxidative stress (Pan et al. 2016; Zhu 2001). Maintaining constant intracellular K+ and Na+ balance is essential for metabolic processes in cells and is crucial for plant adaptation in response to saline environments (Zhu 2003). Previous studies have reported that endophyte infection could adjust Na+ and K+ concentrations in host plants under stress. For example, Bayat et al. (2009) found that Epichloë endophytes increased K+ and Ca2+ contents in F. arundinacea under drought stress. Reza and Mirlohi (2010) showed that E. coenophiala and E. uncinata endophyte infection reduced Na+ and Cl− concentrations in tall fescue and meadow fescue roots, but increased K+ concentrations in the shoots under salt stress. Both Song et al. (2015) and Chen et al. (2018) reported that E. bromicola infection reduced Na+ content, Na+/K+ ratio, and shoot Ca2+ content, but increased K+ content and root Ca2+ content in Hordeum brevisubulatum under salt and alkali stresses. The Na+ content decline in E+ plants led to better plant growth under stresses compared to that in E- plants. Restriction of Na+ transportation and increase in K+ concentration to ensure a high cytosolic K+/Na+ ratio are very important for plants to tolerate high salt levels (Berthomieu et al. 2003; Cuin et al. 2003). These changes could decrease the levels of toxic ions and osmotic influence on plants under stress treatments. Ca2+ is essential for selective ion transport mechanisms and maintenance of K+ influx and Na+/K+ selectivity. In this study, Epichloë endophyte infection did not show significant effects on these indices in most plants.
The effects of stress and mutation on stoichiometry of C, N and P of perennial ryegrass
In the present study, C and P contents decreased under stress, whereas N content increased. C, N, and P are essential elements, and the tissue elemental stoichiometry has a mechanistic linkage with the growth rate of the organism. The growth rate hypothesis proposes that higher growth rates are associated with lower C/N, C/P, and N/P ratios (Hessen et al. 2007). N availability can stimulate phosphatase activity in the roots (Fujita et al. 2010), which could potentially promote P uptake. P is required to meet the protein synthesis demands for increased growth rates (Hessen et al. 2007). Epichloë endophytes also adjusted C, N, and P contents and C/N and C/P ratios to increase host growth (Song et al. 2015; Song et al. 2016; Chen et al. 2018; Xia et al. 2018). For example, Vázquez-de-Aldana et al. (2013) noted that E. festucae alters the nutrient content of F. rubra regardless of water availability. Song et al. (2015) showed that E+ H. brevisubulatum plants had higher contents of N, P and lower ratios of C/N and C/P under salt stress, corroborating the report of Chen et al. (2018). For salt and alkali stresses, Xia et al. (2018) reported that E+ A. inebrians plants had higher N and P contents under soil water deficit. However, endophytes alleviate these changes only in very few cases.
Endophyte-derived alkaloid production is one of the key traits considered by pasture breeders when selecting endophyte strains for pasture grass breeding program. Alkaloids may play some roles in helping endophytes confer protection to plants against abiotic and biotic stresses (Nagabhyru et al. 2013; Schardl et al. 2013). For example, alkaloid levels of E+ A. inebrians increases as NaCl concentration increases, and decreases as water content increases in the soil (Zhang et al. 2011). Nagabhyru et al. (2013) showed that loline alkaloid levels increased in response to drought stress in E+ tall fescue. The chemical ecology mediated by endophytes in grasses has been revealed to be far more complex (Saikkonen et al. 2013). The content variations of N and P contents under stress in our study may also have impacts on alkaloid concentrations, because N is an important component for alkaloid biosynthesis and P availability influences ergot alkaloid production in endophyte-infected grasses (Belesky et al. 1987; Malinowski et al. 1998; Faeth and Fagan 2002). Alkaloid production in these individual plants will be evaluated as soon as possible in the near future to provide a more comprehensive understanding for selection and breeding of these materials.
The effects of space mutation on perennial ryegrass
Space mutations result in abundant and non-directional mutations, which create genetic variability to improve various complicated traits in plants. Space-induced mutation breading is an effective way to breed new varieties and enhance genetic diversity (Liu et al. 2008). In the present study, space mutation had effects on plant performance as space mutation increased tiller number and underground biomass, plant K+ and Ca2+ contents, underground C content, and underground ratio of C/N; however, it reduced plant phytohormone contents, aboveground Na+ content, Na+/K+, and aboveground C/N ratio under some treatments. The mutant individuals provided new methods and resources for perennial ryegrass breeding with strong stress tolerance. Using space-induced radiation, a number of advantageous mutations, which were used to make a breakthrough in most desired crop yield, were also achieved. China has produced 41 varieties developed through space–induced mutation breeding of various crop species such as rice, wheat, cotton, sesame, pepper, tomato, and alfalfa (Liu et al. 2008). However, space-mutation approach results in abundant, non-directional mutations (He et al. 2006). This breeding methodology needs to be followed by many studies, such as material selection, molecular screening of mutants, and earlier generation identification of quality characters to successfully breed new varieties. We should continue to select excellent individual plants from the second generation of these perennial ryegrass germplasms with space mutations and conduct characterisation and genetic analysis in combination with molecular techniques. There are no reports on the effects of space mutations on Epichloë endophytes. In the present study, space mutations did not affect endophyte function. Epichloë endophyte, which lived inside E+ seeds, went through space-induced radiation, possibly having radiation mutations in the genome. Studies on endophyte isolation from E+ mutation plants and its characteristics are in progress to reveal the mutation site and mechanism.