Herbicidal proteins from Bacillus wiedmannii isolate ZT selectively inhibit ryegrass (Lolium temulentum L.).

BACKGROUND: The widespread use of chemical herbicides and the growing issue of weed resistance pose significant challenges in agriculture. To address these problems, there is a pressing need to develop biological herbicides based on bacterial metabolites. RESULTS: In this study, we investigated the impact of the cell-free culture filtrate (CFCF) from the ZT isolate, a bacilliform bacterium obtained from diseased wheat seeds, on the germination and seedling growth of various plant species, including wild oat, ryegrass, redroot, wheat, and chickpea. The results revealed that CFCF had a detrimental effect on the fresh and dry weight of stems and roots in most of the studied plants, except chickpeas. The CFCF was further subjected to separation into aqueous and organic phases using chloroform, followed by the division of the aqueous phase into 13 fractions using an alumina column. Notably, both the aqueous phase (20%) and all 13 fractions (ranging from 50% to 83%) displayed the ability to reduce the root length of ryegrass, a monocotyledonous weed. Liquid chromatography-mass spectrometry (LC-MS) analysis identified that fractions 3 and 7, which were effective against ryegrass but not redroot, contained Cry family proteins, including Cry10 Aa, Cry4 Ba, and Cry4 Aa. Additionally, 16s rRNA gene sequencing revealed that the ZT isolate is closely related (98.27%) to Bacillus wiedmannii. CONCLUSION: Conclusively, metabolites from the ZT bacterium hold promise for monocotyledonous weed-targeted herbicides, providing a constructive strategy to confront agricultural issues tied to chemical herbicides and weed resistance. © 2024 Society of Chemical Industry.

Anti-herbivory defences delivered by Epichloë fungal endophytes: a quantitative review of alkaloid concentration variation among hosts and plant parts.

BACKGROUND AND AIMS: In the subfamily Poöideae (Poaceae), certain grass species possess anti-herbivore alkaloids synthesized by fungal endophytes that belong to the genus Epichloë (Clavicipitaceae). The protective role of these symbiotic endophytes can vary, depending on alkaloid concentrations within specific plant-endophyte associations and plant parts. METHODS: We conducted a literature review to identify articles containing alkaloid concentration data for various plant parts in six important pasture species, Lolium arundinaceum, Lolium perenne, Lolium pratense, Lolium multiflorum|Lolium rigidum and Festuca rubra, associated with their common endophytes. We considered the alkaloids lolines (1-aminopyrrolizidines), peramine (pyrrolopyrazines), ergovaline (ergot alkaloids) and lolitrem B (indole-diterpenes). While all these alkaloids have shown bioactivity against insect herbivores, ergovaline and lolitrem B are harmful for mammals. KEY RESULTS: Loline alkaloid levels were higher in the perennial grasses L. pratense and L. arundinaceum compared to the annual species L. multiflorum and L. rigidum, and higher in reproductive tissues than in vegetative structures. This is probably due to the greater biomass accumulation in perennial species that can result in higher endophyte mycelial biomass. Peramine concentrations were higher in L. perenne than in L. arundinaceum and not affected by plant part. This can be attributed to the high within-plant mobility of peramine. Ergovaline and lolitrem B, both hydrophobic compounds, were associated with plant parts where fungal mycelium is usually present, and their concentrations were higher in plant reproductive tissues. Only loline alkaloid data were sufficient for below-ground tissue analyses and concentrations were lower than in above-ground parts. CONCLUSIONS: Our study provides a comprehensive synthesis of fungal alkaloid variation across host grasses and plant parts, essential for understanding the endophyte-conferred defence extent. The patterns can be understood by considering endophyte growth within the plant and alkaloid mobility. Our study identifies research gaps, including the limited documentation of alkaloid presence in roots and the need to investigate the influence of different environmental conditions.

High-resolution bulked segregant analysis enables candidate gene identification for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.).

Bacterial wilt, caused by Xanthomonas translucens pv. graminis (Xtg), is a serious disease of economically important forage grasses, including Italian ryegrass (Lolium multiflorum Lam.). A major QTL for resistance to Xtg was previously identified, but the precise location as well as the genetic factors underlying the resistance are yet to be determined. To this end, we applied a bulked segregant analysis (BSA) approach, using whole-genome deep sequencing of pools of the most resistant and most susceptible individuals of a large (n = 7484) biparental F2 population segregating for resistance to Xtg. Using chromosome-level genome assemblies as references, we were able to define a ~300 kb region highly associated with resistance on pseudo-chromosome 4. Further investigation of this region revealed multiple genes with a known role in disease resistance, including genes encoding for Pik2-like disease resistance proteins, cysteine-rich kinases, and RGA4- and RGA5-like disease resistance proteins. Investigation of allele frequencies in the pools and comparative genome analysis in the grandparents of the F2 population revealed that some of these genes contain variants with allele frequencies that correspond to the expected heterozygosity in the resistant grandparent. This study emphasizes the efficacy of combining BSA studies in very large populations with whole genome deep sequencing and high-quality genome assemblies to pinpoint regions associated with a binary trait of interest and accurately define a small set of candidate genes. Furthermore, markers identified in this region hold significant potential for marker-assisted breeding strategies to breed resistance to Xtg in Italian ryegrass cultivars more efficiently.

High-affinity iron uptake is required for optimal Epichloë festucae colonization of Lolium perenne and seed transmission.

Epichloë festucae uses a siderophore-mediated system to acquire iron, which is important to maintain endophyte-grass symbioses. Here we investigate the roles of the alternative iron acquisition system, reductive iron assimilation (RIA), via disruption of the fetC gene, which encodes a multicopper ferroxidase, either alone (i.e., ΔfetC) or in combination with disruption of the gene sidA, which encodes a siderophore biosynthesis enzyme (i.e., ΔfetC/ΔsidA). The phenotypic characteristics of these mutants were compared to ΔsidA and wild-type (WT) strains during growth under axenic culture conditions (in culture) and in symbiosis with the host grass, perennial ryegrass (in planta). Under iron deficiency, the colony growth rate of ΔfetC was slightly slower than that of WT, while the growth of ΔsidA and ΔfetC/ΔsidA mutants was severely suppressed. Siderophore analyses indicated that ΔfetC mutants hyperaccumulate ferriepichloënin A (FEA) at low iron concentrations and ferricrocin and FEA at higher iron concentrations. When compared to WT, all mutant strains displayed hyperbranching hyphal structures and a reduced ratio of Epichloë DNA to total DNA in planta. Furthermore, host colonization and vertical transmission through infection of the host seed were significantly reduced in the ΔfetC/ΔsidA mutants, confirming that high-affinity iron uptake is a critical process for Epichloë transmission. Thus, RIA and siderophore iron uptake are complementary systems required for the maintenance of iron metabolism, fungal growth, and symbiosis between E. festucae and perennial ryegrass.

Host Genetic Background Effect on Vertical Seed Transmission of Epichloë Endophyte Strains in Tall Fescue.

Tall fescue (Lolium arundinaceum (Schreb.) Darbysh.) is a cool-season perennial grass widely grown for forage and turf. Tall fescue lives in association with a fungal endophyte that helps the grass overcome abiotic and biotic stressors. The endophyte is asexual and transmits vertically from the tall fescue plant to the next generation through the seed. Producers of endophyte-infected tall fescue must have endophyte infection in at least 70% of their seed. Therefore, endophyte seed transmission is vital in breeding and seed production. Transfer of endophytes from their native host to different backgrounds of elite tall fescue cultivars can lead to a low seed transmission of the endophyte to the seed. This study screened 23 previously uncharacterized endophyte strains for transmissibility when artificially inoculated into continental and Mediterranean-type host tall fescue. We found no correlation between the rate of successful inoculation and the seed transmission rate of the endophyte in the new host. Nor did the seed transmission rate of the endophyte strains in their native host correlate with the seed transmission rate of the endophyte in the new host. Five strains exhibited seed transmission above 70% in both Mediterranean and Continental host backgrounds and will be characterized further for potential use in cultivar development.

Assessing the impact of phyllosphere microbiota on dynamics of in-silo fermentation of Italian ryegrass harvested at heading and blooming stages.

BACKGROUND: The present study aimed to investigate the relationship between the phyllosphere microbiota of Italian ryegrass (Lolium multiflorum Lam.) harvested at heading (H) [> 50% earing rate or 216 g kg-1 fresh weight (FW)] and blooming (B) (> 50% bloom or 254 g kg-1 FW) stages and in-silo fermentation products, and the composition, abundance, diversity and activity of bacterial community. In total, 72 (4 treatments × 6 ensiling durations × 3 replicates) laboratory scale (400 g) silages of Italian ryegrass were prepared: (i) irradiated heading stage silages (IRH) (n = 36) were inoculated with phyllosphere microbiota inoculum (2 mL) eluted from fresh Italian ryegrass at either heading (IH) (n = 18) or blooming (IB) (n = 18) stages; (ii) irradiated blooming stage silages (IRB) (n = 36) were inoculated with either IH (n = 18) or IB (n = 18). Triplicate silos of each treatment were analyzed after 1, 3, 7, 15, 30 and 60 days of ensiling. RESULTS: In fresh forage, Enterobacter, Exiguobacterium and Pantoea were the three major genera at heading stage, and Rhizobium, Weissella and Lactococcus were the most abundant genera at blooming stage. Higher metabolic activity was found in IB. After 3 days of ensiling, the large amounts of lactic acid in IRH-IB and IRB-IB can be attributed to the higher abundances of Pediococcus and Lactobacillus, 1-phosphofructokinase, fructokinase, l-lactate dehydrogenase and glycolysis I, II and III. CONCLUSION: The composition, abundance, diversity and functionality of the phyllosphere microbiota of Italian ryegrass at different growth stages could remarkably affect silage fermentation characteristics. © 2023 Society of Chemical Industry.

Differences in the Characteristics and Pathogenicity of Pyrenophora Species Associated with Seeds of Italian Ryegrass.

Pyrenophora is a genus of pathogens that cause leaf damage and a common seedborne fungus of Italian ryegrass (Lolium multiflorum Lam.). To elucidate the main seedborne Pyrenophora species, 36 seed samples of Italian ryegrass were collected; in total, 113 strains, representing 12.3% of all isolated strains (921), were identified as Pyrenophora species using the identity of ITS sequences in NCBI and the similarity of morphological characteristics. P. dictyoides (97) was the most frequent species. By pure culture technique, 24 representative pure isolates were obtained for further study. Based on DNA analysis of multiple loci (ITS, LSU, GPDH, CHS-1, and RPB1) and morphological characters, eight Pyrenophora species were identified, P. avenicola, P. chaetomioides, P. dictyoides, P. lolii, P. nobleae, P. teres, P. triseptata, and P. tritici-repentis; among them, P. avenicola, P. tritici-repentis, and P. triseptata were newly reported on Italian ryegrass worldwide. Seed inoculation showed that P. dictyoides, P. lolii, and P. teres remarkably decreased the final germination percentages and germination indexes compared with control treatments (P ≤ 0.05); and plant inoculation showed that P. dictyoides, P. lolii, and P. nobleae could cause typical brown spot in vivo with a higher infection rate (P ≤ 0.05). In conclusion, pathogenicity tests showed that all Pyrenophora species could both inhibit seed germination and infect Italian ryegrass to different degrees; among them, P. dictyoides was the most important seedborne pathogen based on the combination of its isolation and infection rate, followed by P. lolii and P. nobleae. The data generated in this study are helpful for the accurate identification of Pyrenophora species and the development of seedborne disease management strategies.

Transcriptome analysis of Epichloë strains in tall fescue in response to drought stress.

Epichloë coenophiala, a systemic fungal symbiont (endophyte) of tall fescue (Lolium arundinaceum), has been documented to confer to this grass better persistence than plants lacking the endophyte, especially under stress conditions such as drought. The response, if any, of the endophyte to imposition of stress on the host plant has not been characterized previously. Therefore, we investigated effects on gene expression by E. coenophiala and a related endophyte when plant-endophyte symbiota were subjected to acute water-deficit stress. Plants harboring different endophyte strains were grown in sand in the greenhouse, then half were deprived of water for 48 h and the other half were watered controls. RNA was isolated from different plant tissues, and mRNA sequencing (RNA-seq) was conducted to identify genes that were differentially expressed comparing stress treatment with control. We compared two different plants harboring the common toxic E. coenophiala strain (CTE) and two non-ergot-alkaloid-producing Epichloë strains in tall fescue pseudostems, and in a second experiment we compared responses of E. coenophiala CTE in plant pseudostem and crown tissues. The endophytes responded to the stress with increased expression of genes involved in oxidative stress response, oxygen radical detoxification, C-compound carbohydrate metabolism, heat shock, and cellular transport pathways. The magnitude of fungal gene responses during stress varied among plant-endophyte symbiota. Responses in pseudostems and crowns involved some common pathways as well as some tissue-specific pathways. The fungal response to water-deficit stress involved gene expression changes in similar pathways that have been documented for plant stress responses, indicating that Epichloë spp. and their host plants either coordinate stress responses or separately activate similar stress response mechanisms that work together for mutual protection.

Integrative interactomics applied to bovine fescue toxicosis.

Bovine fescue toxicosis (FT) is caused by grazing ergot alkaloid-producing endophyte (Epichloë coenophiala)-infected tall fescue. Endophyte's effects on the animal's microbiota and metabolism were investigated recently, but its effects in planta or on the plant-animal interactions have not been considered. We examined multi-compartment microbiota-metabolome perturbations using multi-'omics (16S and ITS2 sequencing, plus untargeted metabolomics) in Angus steers grazing non-toxic (Max-Q) or toxic (E+) tall fescue for 28 days and in E+ plants. E+ altered the plant/animal microbiota, decreasing most ruminal fungi, with mixed effects on rumen bacteria and fecal microbiota. Metabolic perturbations occurred in all matrices, with some plant-animal overlap (e.g., Vitamin B6 metabolism). Integrative interactomics revealed unique E+ network constituents. Only E+ had ruminal solids OTUs within the network and fecal fungal OTUs in E+ had unique taxa (e.g., Anaeromyces). Three E+-unique urinary metabolites that could be potential biomarkers of FT and targeted therapeutically were identified.

Inbred progeny of tall fescue infected with Epichloë fungal endophyte have a high defence compound content and no depression in seed set.

Substantial evidence is available on the capacity of particular strains of Epichloë fungal endophyte to remove the barriers of self-pollination in host grasses. We hypothesized that this might open up new methods to obtain inbred lines for specific turf hybrids and genetic studies. In the present study, we evaluated the first generation of self-pollination derived plants of endophyte infected clones (EIS1 ) from putative genotypes 75B+ and 75C+ and those from the first generation of self-pollination in six commercial tall fescue clones plus their hybrids with 75B- and 75C- for growth, seed yield and polyphenolic content as an index for biosynthesis of defence compounds under field conditions. The results showed that EIS1 had high hyphal density within leaf sheaths and higher growth and seed-related traits in at least one genotype. There were higher amounts of flavonoid and phenolic compounds (up to twofold) in both genotypes than in their hybrid counterparts and endophyte-free progeny. Selected genotypes within EIS1 contained significantly more chlorogenic acid, p-coumaric acid and rutin than the best non-infected genotypes. We conclude that phenotypic selection of individuals from the S1 population is feasible for improving fitness and stress resistance in novel inbred lines of tall fescue for development of new turf cultivars with the desired ecophysiological traits.

Exploring interactions between Beauveria and Metarhizium strains through co-inoculation and responses of perennial ryegrass in a one-year trial.

Perennial ryegrass (Lolium perenne L.) possesses a high level of nutritional quality and is widely used as a forage species to establish permanent pastures in southern Chile. However, the productivity of most such pastures is limited by various environmental agents, such as insect pests and drought. In this context, our work stresses the need for elucidating the ability of fungal endophytes to establish interactions with plants, and to understand how these processes contribute to plant performance and fitness. Therefore, we evaluated the colonization and impact of two native strains of the endophytic insect-pathogenic fungus (EIPF) group isolated from permanent ryegrass pastures in southern Chile. Roots and seeds of ryegrass and scarabaeid larvae were collected from nine different ryegrass pastures in the Los Ríos region of southern Chile to specifically isolate EIPFs belonging to the genera Beauveria and Metarhizium. Fungal isolations were made on 2% water agar with antibiotics, and strains were identified by analyzing the entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region. Four strains of Beauveria and 33 strains of Metarhizium were isolated only in scarabaeid larvae from ryegrass pastures across four sites. Experimental mini-pastures that were either not inoculated (control) or co-inoculated with conidia of the strains Beauveria vermiconia NRRL B-67993 (P55_1) and Metarhizium aff. lepidiotae NRRL B-67994 (M25_2) under two soil humidity levels were used. Ryegrass plants were randomly collected from the mini-pastures to characterize EIPF colonization in the roots by real-time PCR and fluorescence microscopy. Aboveground biomass was measured to analyze the putative impact of colonization on the mini-pastures' aboveground phenotypic traits with R software using a linear mixed-effects model and the ANOVA statistical test. Seasonal variation in the relative abundance of EIPFs was observed, which was similar between both strains from autumn to spring, but different in summer. In summer, the relative abundance of both EIPFs decreased under normal moisture conditions, but it did not differ significantly under water stress. The aboveground biomass of ryegrass also increased from autumn to spring and decreased in summer in both the inoculated and control mini-pastures. Although differences were observed between moisture levels, they were not significant between the control and inoculated mini-pastures, except in July (fresh weight and leaf area) and October (dry weight). Our findings indicate that native strains of B. vermiconia NRRL B-67993 (P55_1) and M. aff. lepidiotae NRRL B-67994 (M25_2) colonize and co-exist in the roots of ryegrass, and these had little or no effect on the mini-pastures' aboveground biomass; however, they could have other functions, such as protection against root herbivory by insect pests.

Insights into soil bacterial and physicochemical properties of annual ryegrass-maize rotation (ARMR) system in southern China.

The popularized application of annual ryegrass-maize rotation (ARMR) in southern China has been proposed to fully utilize the farmlands and to increase forage yield and quality. Herein, one growth cycle of ARMR was conducted and soil bacteria were analyzed by 16S rRNA sequencing for control (CK), after the preceding crop (monoculture, or mixed sowing of annual ryegrass and oat) and the successive crop (maize). Our results indicated that the α-diversity of soil bacteria was changed in the ARMR system, which was related to the activity of urease and available phosphatase. The mixed sowing of annual ryegrass and oat in preceding crop could improve the yield and quality, while it was accompanied by unbalanced soil community. With the increased sowing proportion of oat to annual ryegrass, the soil pH increased while the soil available phosphatase decreased. The ARMR system was found to benefit the soil microenvironment by increasing the beneficial soil bacteria and enzyme activity or decreasing the harmful soil bacteria. Considering the soil bacteria α-diversity index and physicochemical properties comprehensively, the recommended sowing regime is the mixed sowing of M2 (22.5 kg·hm-2 annual ryegrass with 75 kg·hm-2 oat).

Growth-promoting effects of Bradyrhizobium soybean symbionts in black oats, white oats, and ryegrass.

Although inoculating soybean with rhizobia for biological nitrogen fixation is a common practice in agriculture, rhizobia are also known to associate with grasses. In this study, we evaluate the potential utility of the rhizobial strains SEMIA 587 and 5019 (Bradyrhizobium elkanii), 5079 (Bradyrhizobium japonicum), and 5080 (Bradyrhizobium diazoefficiens), recommended for Brazilian soybean inoculation, in colonizing black oat plants and promoting growth in black and white oats, and ryegrass. Inoculation of white oats with SEMIA 587 increase the seed germination (SG) by 32.09%, whereas the SG of black oats inoculated with SEMIA 587 and 5019 increased by 40.38% and 37.85%, respectively. Similarly, inoculation of ryegrass with all strains increased SG values between 24.63 and 27.59%. In addition, white oats with SEMIA 587 and 5080 had root areas significantly superior to those in other treatments, whereas inoculation with SEMIA 5079 and 5080 resulted in the highest volume of roots. Likewise, SEMIA 5079 and 5080 significantly increased the length, volume, and area of black oats roots, whereas SEMIA 587 increased the volume, area, and dry mass of roots and shoot. Inoculation in ryegrass with SEMIA 587 significantly increased the root volume. Moreover, most strains transformed with gfp and gus were observed to colonize the roots of black oats. Collectively, the findings of this study indicate that rhizobial strains recommended for inoculation of soybean can also be used to promote the growth of the three assessed grass species, and are able to colonize the roots of black oats.

Effects of Tall Fescue Endophyte Type and Dopamine Receptor D2 Genotype on Cow-Calf Performance during Late Gestation and Early Lactation.

Grazing endophyte-infected, toxic tall fescue reduces cow/calf production; therefore, this study examines alternate strategies such as use of novel endophyte fescue varieties during late gestation and early lactation or genetic selection of resistant cows. Pregnant cows (n = 75) were randomly assigned to fescue endophyte type: 1) endophyte-infected ergot alkaloid producing tall fescue (E+) or 2) novel endophyte-infected, non-toxic tall fescue (NOV) within maternal (A|A, n = 38 and G|G, n = 37) DRD2 genotype to examine changes in cow/calf performance and milk production during late gestation and early lactation. Grazing E+ fescue pastures during late gestation reduced cow body weight gain but did not alter calf birth weight compared to NOV. Milk production and calf ADG during the first 30 day of lactation were lower for E+ than NOV. The calving rate was reduced, but not calving interval for E+ cows. The adjusted 205-day weight of calves was lower in those grazing E+ with their dams compared to NOV. There were no interactions between DRD2 genotype and fescue endophyte type indicating that genotype was not associated with response to E+ fescue in this study. Overall, grazing NOV tall fescue pastures rather than E+ during critical stages of production improved cow gain during late gestation, calving rate, early milk production and calf growth.

Arbuscular mycorrhizal fungus facilitates ryegrass (Lolium perenne L.) growth and polychlorinated biphenyls degradation in a soil applied with nanoscale zero-valent iron.

Nanoscale zero-valent iron (nZVI) shows an excellent degradation effect on chlorinated contaminants in soil, but poses a threat to plants in combination with phytoremediation. Arbuscular mycorrhizal (AM) fungus can reduce the phyototoxicity of nZVI, but their combined impacts on polychlorinated biphenyls (PCBs) degradation and plant growth remain unclear. Here, a greenhouse pot experiment was conducted to investigate the influences of nZVI and/or Funneliformis caledonium on soil PCB degradation and ryegrass (Lolium perenne L.) antioxidative responses. The amendment of nZVI significantly reduced not only the total and homolog concentrations of PCBs in the soil, but also the ryegrass biomass as well as soil available P and root P concentrations. Moreover, nZVI significantly decreased leaf superoxide disutase (SOD) activity, while tended to decrease the protein content. In contrast, the additional inoculation of F. caledonium significantly increased leaf SOD activity and protein content, while tended to increase the catalase activity and tended to decrease the malondialdehyde content. The additional inoculation of F. caledonium also significantly increased soil alkaline phosphatase activity, and tended to increase root P concentration, but had no significantly effects on soil available P concentration, the biomass and P acquisition of ryegrass, which could be attributed to the fixation of soil available nutrients by nZVI. Additionally, F. caledonium facilitated PCB degradation in the nZVI-applied soil. Thus, AM fungus can alleviate the nZVI-induced phytotoxicity, showing great application potentials in accompany with nZVI for soil remediation.

Getting ready for the ozone battle: Vertically transmitted fungal endophytes have transgenerational positive effects in plants.

Ground-level ozone is a global air pollutant with high toxicity and represents a threat to plants and microorganisms. Although beneficial microorganisms can improve host performance, their role in connecting environmentally induced maternal plant phenotypes to progeny (transgenerational effects [TGE]) is unknown. We evaluated fungal endophyte-mediated consequences of maternal plant exposure to ozone on performance of the progeny under contrasting scenarios of the same factor (high and low) at two stages: seedling and young plant. With no variation in biomass, maternal ozone-induced oxidative damage in the progeny that was lower in endophyte-symbiotic plants. This correlated with an endophyte-mediated higher concentration of proline, a defence compound associated with stress control. Interestingly, ozone-induced TGE was not associated with reductions in plant survival. On the contrary, there was an overall positive effect on seedling survival in the presence of endophytes. The positive effect of maternal ozone increasing young plant survival was irrespective of symbiosis and only expressed under high ozone condition. Our study shows that hereditary microorganisms can modulate the capacity of plants to transgenerationally adjust progeny phenotype to atmospheric change.

The Impact of Alkaloid-Producing Epichloë Endophyte on Forage Ryegrass Breeding: A New Zealand Perspective.

For 30 years, forage ryegrass breeding has known that the germplasm may contain a maternally inherited symbiotic Epichloë endophyte. These endophytes produce a suite of secondary alkaloid compounds, dependent upon strain. Many produce ergot and other alkaloids, which are associated with both insect deterrence and livestock health issues. The levels of alkaloids and other endophyte characteristics are influenced by strain, host germplasm, and environmental conditions. Some strains in the right host germplasm can confer an advantage over biotic and abiotic stressors, thus acting as a maternally inherited desirable 'trait'. Through seed production, these mutualistic endophytes do not transmit into 100% of the crop seed and are less vigorous than the grass seed itself. This causes stability and longevity issues for seed production and storage should the 'trait' be desired in the germplasm. This makes understanding the precise nature of the relationship vitally important to the plant breeder. These Epichloë endophytes cannot be 'bred' in the conventional sense, as they are asexual. Instead, the breeder may modulate endophyte characteristics through selection of host germplasm, a sort of breeding by proxy. This article explores, from a forage seed company perspective, the issues that endophyte characteristics and breeding them by proxy have on ryegrass breeding, and outlines the methods used to assess the 'trait', and the application of these through the breeding, production, and deployment processes. Finally, this article investigates opportunities for enhancing the utilisation of alkaloid-producing endophytes within pastures, with a focus on balancing alkaloid levels to further enhance pest deterrence and improving livestock outcomes.

Genetic Manipulation of the Ergot Alkaloid Pathway in Epichloë festucae var. lolii and Its Effect on Black Beetle Feeding Deterrence.

Epichloë endophytes are filamentous fungi (family Clavicipitaceae) that live in symbiotic associations with grasses in the sub family Poöideae. In New Zealand, E. festucae var. lolii confers significant resistance to perennial ryegrass (Lolium perenne) against insect and animal herbivory and is an essential component of pastoral agriculture, where ryegrass is a major forage species. The fungus produces in planta a range of bioactive secondary metabolites, including ergovaline, which has demonstrated bioactivity against the important pasture pest black beetle, but can also cause mammalian toxicosis. We genetically modified E. festucae var. lolii strain AR5 to eliminate key enzymatic steps in the ergovaline pathway to determine if intermediate ergot alkaloid compounds can still provide insecticidal benefits in the absence of the toxic end product ergovaline. Four genes (dmaW, easG, cloA, and lpsB) spanning the pathway were deleted and each deletion mutant was inoculated into five different plant genotypes of perennial ryegrass, which were later harvested for a full chemical analysis of the ergot alkaloid compounds produced. These associations were also used in a black beetle feeding deterrence study. Deterrence was seen with just chanoclavine present, but was cumulative as more intermediate compounds in the pathway were made available. Ergovaline was not detected in any of the deletion associations, indicating that bioactivity towards black beetle can be obtained in the absence of this mammalian toxin.

Genome-Wide Association Mapping of Crown and Brown Rust Resistance in Perennial Ryegrass.

A population of 239 perennial ryegrass (Lolium perenne L.) genotypes was analyzed to identify marker-trait associations for crown rust (Puccinia coronata f. sp. lolii) and brown rust (Puccinia graminis f. sp. loliina) resistance. Phenotypic data from field trials showed a low correlation (r = 0.17) between the two traits. Genotypes were resequenced, and a total of 14,538,978 SNPs were used to analyze population structure, linkage disequilibrium (LD), and for genome-wide association study. The SNP heritability (h2SNP) was 0.4 and 0.8 for crown and brown rust resistance, respectively. The high-density SNP dataset allowed us to estimate LD decay with the highest possible precision to date for perennial ryegrass. Results showed a low LD extension with a rapid decay of r2 value below 0.2 after 520 bp on average. Additionally, QTL regions for both traits were detected, as well as candidate genes by applying Genome Complex Trait Analysis and Multi-marker Analysis of GenoMic Annotation. Moreover, two significant genes, LpPc6 and LpPl6, were identified for crown and brown rust resistance, respectively, when SNPs were aggregated to the gene level. The two candidate genes encode proteins with phosphatase activity, which putatively can be induced by the host to perceive, amplify and transfer signals to downstream components, thus activating a plant defense response.

Biostimulating effect of l-tryptophan on the yield and chemical and microbiological quality of perennial ryegrass (Lolium perenne) herbage and silage for ruminant.

BACKGROUND: This study aimed to evaluate the effect of l-tryptophan (l-TRP) used in the cultivation of Lolium perenne on the yield, and chemical and microbiological quality of its herbage and silage. l-Tryptophan was applied in doses of 5, 0.5, 0.05 kg ha-1 . The experiment was conducted with a control group (C) and a comparative control group (C+) with higher nitrogen fertilization. RESULTS: The dose of 5 kg ha-1 had a significant effect on herbage yield, which increased by 15% compared to group C and approximated the value achieved in group C+. The treatment with l-TRP caused a significant increase in water-soluble carbohydrate (WSC) content only in Tr5 (165 g kg-1 DM), which was reflected in a more beneficial course of fermentation, lower pH (4.59), and a higher sum of fermentation acids, including lactic acid (94.7 g kg-1 DM). Ryegrass treatment with a high l-TRP dose effectively reduced the loads of Clostridium spp. and fungi, and increased the count of Bacillus spp. The l-TRP significantly reduced N-NH3 content in Tr05 (98.6 g kg-1 TN) compared with C+ (123 g kg-1 TN) and butyric acid content in Tr05 (from 1.35 g kg-1 DM in the C to 0.38 g kg-1 DM). CONCLUSION: The most effective dose turned out to be a dose of 5 kg ha-1 , which allowed a higher yield and a better fermentation course to be achieved. This work presents the feasibility of using l-TRP to optimize nutrient consumption by Lolium perenne and ultimately to affect the quality of its silage as a feedstuff. © 2020 Society of Chemical Industry.