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.

Multi-species pastures for grazing dairy cows in small-scale dairy systems in the highlands of Mexico.

Grazing in small-scale dairy systems reduces costs and enhances sustainability. However, binary pastures (BP) have low persistency, which may be increased by including more species given their flexibility to withstand different agroecological and management situations. The objective was to assess a multi-species (MS) pasture of perennial ryegrass (RG), tall fescue (TF), bromegrass (BG), red clover (RC) and white clover (WC) in comparison to a BP of perennial ryegrass (RG) - WC grazed by six Holstein dairy cows during the dry season in an on-farm double cross-over experiment, with three 14-day each experimental periods for animal variables, and a split-plot design for pasture variables at 3 cow/ha. There were differences (P < 0.05) between pastures for sward height (MS 5.0 vs. BP 4.0 ± 0.10 cm, both MS and BP) and net herbage accumulation (MS 902 vs BP 228 ± 172.4 kg DM/ha, both MS and BP) and differences (P < 0.05) for chemical composition among periods. There were differences (P < 0.01) among periods for estimated DM intake, but no differences (P > 0.05) for milk yield (mean 16.8 kg/cow/day), milk fat or protein content (mean 31.8 and 28.8 g/kg). Multi-species pastures are a viable alternative for SSDS in the highlands of central Mexico, being more resistant to harsh environment and to weed and kikuyu grass invasion.

Accumulation of Target Gene Mutations Confers Multiple Resistance to ALS, ACCase, and EPSPS Inhibitors in Lolium Species in Chile.

Different Lolium species, common weeds in cereal fields and fruit orchards in Chile, were reported showing isolated resistance to the acetyl CoA carboxylase (ACCase), acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibiting herbicides in the late 1990s. The first case of multiple resistance to these herbicides was Lolium multiflorum found in spring barley in 2007. We hypothesized that other Lolium species may have evolved multiple resistance. In this study, we characterized the multiple resistance to glyphosate, diclofop-methyl and iodosulfuron-methyl-sodium in Lolium rigidum, Lolium perenne and Lolium multiflorum resistant (R) populations from Chile collected in cereal fields. Lolium spp. populations were confirmed by AFLP analysis to be L. rigidum, L. perenne and L. multiflorum. Dose-response assays confirmed multiple resistance to glyphosate, diclofop-methyl and iodosulfuron methyl-sodium in the three species. Enzyme activity assays (ACCase, ALS and EPSPS) suggested that the multiple resistance of the three Lolium spp. was caused by target site mechanisms, except the resistance to iodosulfuron in the R L. perenne population. The target site genes sequencing revealed that the R L. multiflorum population presented the Pro-106-Ser/Ala (EPSPS), Ile-2041-Asn++Asp-2078-Gly (ACCase), and Trp-574-Leu (ALS) mutations; and the R L. rigidum population had the Pro-106-Ser (EPSPS), Ile-1781-Leu+Asp-2078-Gly (ACCase) and Pro-197-Ser/Gln+Trp-574-Leu (ALS) mutations. Alternatively, the R L. perenne population showed only the Asp-2078-Gly (ACCase) mutation, while glyphosate resistance could be due to EPSPS gene amplification (no mutations but high basal enzyme activity), whereas iodosulfuron resistance presumably could involve non-target site resistance (NTSR) mechanisms. These results support that the accumulation of target site mutations confers multiple resistance to the ACCase, ALS and EPSPS inhibitors in L. multiflorum and L. rigidum from Chile, while in L. perenne, both target and NTSR could be present. Multiple resistance to three herbicide groups in three different species of the genus Lolium in South America represents a significant management challenge.

Fructan and antioxidant metabolisms in plants of Lolium perenne under drought are modulated by exogenous nitric oxide.

Drought is a major environmental factor that can trigger oxidative stress and affect plant growth and productivity. Previous studies have shown that exogenous nitric oxide (NO) can minimize oxidative stress-related damage through the modulation of antioxidant enzyme activity. Fructan accumulation also has an important role in drought tolerance, since these carbohydrates participate in osmoregulation, membrane protection and oxidant scavenging. Currently, there are few studies investigating NO-regulated fructan metabolism in response to abiotic stresses. In the present study, we sought to determine if treating plants of Lolium perenne with S-nitrosoglutathione (GSNO), a NO donor, improved drought tolerance. Two-month-old plants received water (control), GSNO and reduced glutathione (GSH) as foliar spray treatments and were then maintained under drought or well-watered conditions for 23 days. At the end of drought period, we evaluated growth, pigment content and antioxidant and fructan metabolisms. None of these conditions influenced dry mass accumulation, but the leaves of plants treated with GSNO exhibited a slight increase in pigment content under drought. GSNO treatment also induced 1-SST activity, which was associated with a 3-fold increase in fructan content. GSNO-treated plants presented higher GR activity and, consequently, increased GSH levels. L. perenne cv. AberAvon was relatively tolerant to the water stress condition employed herein, maintaining ROS homeostasis and mitigating oxidative stress, possibly due to fructan, ascorbate and glutathione pools.

Milk Production, Milk Quality, and Behaviour of Dairy Cows Grazing on Swards with Low and High Water-Soluble Carbohydrates Content in Autumn: A Pilot Trial.

Grazing ruminant systems can be sustainably intensified by improving efficiency while reducing their environmental impact. The objective of the present study was to examine the potential of pastures differing in water-soluble carbohydrates (WSC) and crude protein (CP) contents to affect milk production and composition as well as the behaviour of cows grazing perennial ryegrass (PRG) swards. By modifying the nitrogen (N) fertilisation rate (83 and 250 kg/ha per year) and the defoliation frequency (two or three leaves per tiller) in combination with cultivar selection (high-sugar vs. standard cultivars), we obtained two swards differing in WSC and CP contents. The two contrasting swards were each grazed by six dairy cows in nine daily strips in autumn. Pasture samples were collected to determine herbage mass and quality. Cow behaviour was recorded by direct observation. Herbage offered and apparently consumed were similar between swards (averaging 37.3 and 18.2 kg/cow, respectively), although the residual was lower in the high-sugar sward (1735 vs. 2143 kg/ha). Cows spent less time grazing in the high-sugar sward (66.9% v. 71.6%), but the rumination times was similar (14.6%). Milk production and composition were similar between groups, suggesting that high-quality pastures would require a greater difference in nutritional composition to affect animal performance.