First Report of Brown Leaf Spot of Elymus nutans Caused by Bipolaris sorokiniana in Northwestern China.

Elymus nutans is an important feed and forage resource in alpine region of northwestern China, and helps to maintain the ecological balance on Qinghai-Tibetan plateau (Fei et al. 2010). In September 2021, brown leaf spots were observed in Shandan Horse Farm (38.3061°N, 101.2453°E), Gansu province of China.Disease severity of leaves was estimated to be 38% based on a random sample of 100 leaves from each of 10 arbitrarily placed quadrats on the farm.The typical symptoms on leaves appeared as brown to dark brown spots with grayish white in the center, surrounded by a yellow halo. A total of twenty diseased leaves were collected from five different plants in the field. The lesions (about 5 mm) from these samples were initially soaked in 75% ethanol for 30 s followed by soaking in 1% NaClO solution for 1 min, repeatedly rinsed with sterilized water, air dried, and put on potato dextrose agar (PDA). After 7 days of incubation at 25°C in the dark, the same fungus was consistently isolated; the pure isolates were obtained by single-spore cultures (Cai et al. 2009). The colonies were irregular in shape and black in color with white margins. Conidia were spindle-shaped, dark brown, 5 to 9 septa (7 septa in general), and a size range of 21.74 to 78.97 × 7.95 to 21.12 µm (avg. 57.25 × 17.76 µm, n = 50),morphologically similar to B. sorokiniana reported on Avena nuda in China (Li et al. 2019). The ITS region of rDNA and partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of the representative isolate PJC5 were amplified using the primers ITS1/ITS4 and gpd1/gpd2, respectively (Manamgoda et al. 2014). After sequencing sequences were deposited into GenBank with accession numbers OM419194 (ITS) and OM718009 (GAPDH). BLAST analysis showed 99% and 100% identity with the corresponding sequences (accession numbers MH855159.1 and HG779088.1) of known strain CBS 140.31 of B. sorokiniana (Manamgoda et al. 2014). For pathogenicity tests, ten 8-week-old healthy plants obtained by growing E. nutans in pots were spray-inoculated separately with a conidia suspension of 1 × 106 conidia/ml of isolate PJC5. Ten plants were also sprayed with sterilized distilled water as controls. Then all plants were individually covered with transparent polyethylene bags for three days to maintain high relative humidity and placed in greenhouse at 25°C. Seven days after inoculation, symptoms of leaf infection were similar to those observed in the field, while no symptoms appeared on leaves of control plants. The experiments were conducted three times and the pathogen was re-isolated from inoculated leaves and was confirmed as B. sorokiniana based on morphological and molecular analyses. B. sorokiniana has been reported to cause leaf spot of E. riparius in the United States (Roane et al. 2005) and E. breviaristatus (Zhuang, 2005) and A. nuda (Li et al. 2019) in China. To our knowledge, this is the first report of B. sorokiniana causing leaf spot on E. nutans in China. Identifying this pathogen provides a foundation to prevent this complex disease and to reduce economic loss.

Does Epichloë Endophyte Enhance Host Tolerance to Root Hemiparasite?

Epichloë endophytes have been shown to be mutualistic symbionts of cool-season grasses under most environmental conditions. Although pairwise interactions between hemiparasites and their hosts are heavily affected by host-associated symbiotic microorganisms, little attention has been paid to the effects of microbe-plant interactions, particularly endophytic symbiosis, in studies examining the effects of parasitic plants on host performance. In this study, we performed a greenhouse experiment to examine the effects of hereditary Epichloë endophyte symbiosis on the growth of two host grasses (Stipa purpurea and Elymus tangutorum) in the presence or absence of a facultative root hemiparasite (Pedicularis kansuensis Maxim). We observed parasitism of both hosts by P. kansuensis: when grown with a host plant, the hemiparasite decreased the performance of the host while improving its own biomass and survival rate of the hemiparasite. Parasitized endophyte-infected S. purpurea plants had higher biomass, tillers, root:shoot ratio, and photosynthetic parameters and a lower number of functional haustoria than the endophyte-free S. purpurea conspecifics. By contrast, parasitized endophyte-infected E. tangutorum had a lower biomass, root:shoot ratio, and photosynthetic parameters and a higher number of haustoria and functional haustoria than their endophyte-free counterparts. Our results reveal that the interactions between the endophytes and the host grasses are context dependent and that plant-plant interactions can strongly affect their mutualistic interactions. Endophytes originating from S. purpurea alleviate the host biomass reduction by P. kansuensis and growth depression in the hemiparasite. These findings shed new light on using grass-endophyte symbionts as biocontrol methods for the effective and sustainable management of this weedy hemiparasite.

[Phylogeny of Neotyphodium endophyte from western Chinese Elymus species based on act sequences].

OBJECTIVE: We analyzed phylogenetic relationships between asexual endophytes isolated from western Chinese Elymus species. METHODS: Fifteen act sequences were cloned from asexual endophytes of the western Chinese Elymus species. The phylogenetic tree and network topology structure were constructed using act sequences obtained and Genbank. RESULTS: The act sequences from asexual endophyte were single-copy gene. The western Chinese asexual endophye (Neotyphodium spp.) has a different origination from the North American sexual endophyte ( Epichloë elymi) from Elymus species, but there is a closer relationship between Neotyphodium spp. and Neotyphodium chisosum from North American Achnatherum eminens. Further, endophyte gene-flow exists between sexual Neotyphodium chisosum from North American Achnatherum eminens ( Hap 8) and asexual Neotyphodium sp. from western Chinese Elymus species (Hap 3).

Discovery of Epichloë as novel endophytes of Psathyrostachys lanuginosa in China and their alkaloid profiling.

The Epichloë genus represents a significant group of above-ground endophytes extensively researched for their potential applications in agriculture and ecology. Additionally, Epichloë species synthesize bioactive alkaloids, which generally cause health problems in livestock and have detrimental effects on the performance of insect herbivores. Psathyrostachys lanuginosa serves as a valuable forage grass for livestock owing to its high nutritional value and resilience in adverse environmental conditions. Nevertheless, to date, no reports have documented Epichloë as endophytes of P. lanuginosa. In this study, four strains (PF5, PF9, QG2, and QG4) were isolated and identified through morphological, molecular, and phylogenetic analyses as endophytes of P. lanuginosa. Morphological analysis indicated colony characteristics and conidia features consistent with symbiotic Epichloë, with no significant differences observed in growth rates or conidia dimensions among the four strains. Phylogenetic analysis confirmed all strains as E. bromicola. Additionally, alkaloid biosynthetic genes were detected, revealing differences in the potential synthesis of peramine and indole diterpenoid alkaloids among strains from different geographic origins. However, all four E. bromicola strains exhibited similar potential for synthesizing ergot alkaloids, but not loline alkaloids. Overall, this study identified P. lanuginosa as a novel host for E. bromicola and provided insights into the alkaloid profiles of these strains, laying a solid foundation for the scientific and rational utilization of Epichloë resources.

Endophytic Fungi Regulate HbNHX1 Expression and Ion Balance in Hordeum bogdanii under Alkaline Stress.

Plants cope with abiotic stress in several ways, including by collaborating with microorganisms. Epichloë, an endophytic fungus, has been shown to improve plant tolerance to extreme external environments. Hordeum bogdanii is a known salt-tolerant plant with the potential to improve alkaline lands. NHX1 plays a key role in the transport of ions in the cell and is overexpressed in plants with increased salt tolerance. However, the expression levels of HbNHX1 in Epichloë endophytic fungal symbionts in H. bogdanii have not been elucidated. We used Hordeum bogdanii (E+) with the endophytic fungi Epichloë bromicola and H. bogdanii (E-) without the endophytic fungi and compared the differences in the ion content and HbNHX1 expression between the shoots and roots of E+ and E- plants under alkaline stress. The absorption capacity of both K+ and Na+ of H. bogdanii with endophytic fungi was higher than that without endophytic fungi. In the absence of alkaline stress, endophytic fungi significantly reduced the Cl- content in the host H. bogdanii. Alkaline stress reduced SO42- content in H. bogdanii; however, compared with E-, endophytic fungi increased the content of SO42- in E+ plants. With an increase in the alkaline concentration, the expression of HbNHX1 in the roots of H. bogdanii with endophytic fungus exhibited an upward trend, whereas the expression in the shoots exhibited a downward trend first and then an upward trend. Under 100 mmol·L-1 mixed alkaline stress, the expression of HbNHX1 in E+ was significantly higher than that in E-, indicating that endophytic fungi could increase the Na+ region in vacuoles. The external environment affects the regulation of endophytic fungi in H. bogdanii and that endophytic fungi can play a key role in soil salinization. Therefore, the findings of this study will provide technical support and a theoretical basis for better utilization of endophytic fungi from H. bogdanii in saline land improvement.

Changes in bud bank and their correlation with plant community composition in degraded alpine meadows.

Bud banks are considered a crucial factor in regulating the species composition of grassland communities and maintaining the ecological function of alpine grasslands. However, few studies have paid attention to the dynamic changes of bud banks from undisturbed to severely degraded alpine meadows. Therefore, this study examined the correlations between plant diversity and bud bank traits at different stages of alpine meadows degradation. Grass biomasses and plant diversity were found to be highest in moderately degraded meadows, and sedge biomasses were highest in lightly degraded meadows. Lack of disturbance and moderate disturbance by herbivores increased the bud bank density of alpine meadows. Consistent with the changes in bud bank density, bud bank diversity was highest in undisturbed meadows. The structural equation model indicated that the densities of rhizome and the densities and diversities of tiller buds play crucial roles in facilitating the greater diversity of the plant community. Our findings suggest that the diversities and densities of rhizome and tiller buds in the degradation stages are synchronized with changes in plant diversity, and in the regenerative ability of bud banks, which largely determine the outcome of restoration in degraded meadows. These findings could provide a frame of reference for effectively restoring degraded alpine regions by regenerating bud banks. The potential driving force and renewal capacity of bud banks should be taken into account in restoring the Qinghai-Tibet Plateau's degraded meadow.

Effect of Epichloë Endophyte on the Growth and Carbon Allocation of Its Host Plant Stipa purpurea under Hemiparasitic Root Stress.

Epichloë endophytes not only affect the growth and resistance of their host plants but also confer nutrient benefits to parasitized hosts. In this study, we used Pedicularis kansuensis to parasitize Stipa purpurea, both with and without endophytic fungi, and to establish a parasitic system. In this study, endophytic fungal infection was found to increase the dry weight of the leaf, stem, and leaf sheath, as well as the plant height, root length, tiller number, aboveground biomass, and underground biomass of S. purpurea under root hemiparasitic stress. Meanwhile, the 13C allocation of the leaf sheaths and roots of S. purpurea increased as the density of P. kansuensis increased, while the 13C allocation of the leaf sheaths and roots of E+ S. purpurea was lower than that of E- S. purpurea. The 13C allocation of the stem, leaf sheath, and root of E+ S. purpurea was higher than that of its E- counterpart. Furthermore, the content of photosynthetic 13C and the 13C partition rate of the stems, leaves, roots, and entire plant of S. purpurea and P. kansuensis transferred from S. purpurea increased as the density of P. kansuensis increased. These results will generate new insights into the potential role of symbiotic microorganisms in regulating the interaction between root hemiparasites and their hosts.

Comparison of the effect of temperature and water potential on the seed germination of five Pedicularis kansuensis populations from the Qinghai-Tibet plateau.

Temperature and water potentials are considered the most critical environmental factors in seed germinability and subsequent seedling establishment. The thermal and water requirements for germination are species-specific and vary with the environment in which seeds mature from the maternal plants. Pedicularis kansuensis is a root hemiparasitic weed that grows extensively in the Qinghai-Tibet Plateau's degraded grasslands and has seriously harmed the grasslands ecosystem and its utilization. Information about temperatures and water thresholds in P. kansuensis seed germination among different populations is useful to predicting and managing the weed's distribution in degraded grasslands. The present study evaluated the effects of temperature and water potentials on P. kansuensis seed germination in cool and warm habitats, based on thermal time and hydrotime models. The results indicate that seeds from cool habitats have a higher base temperature than those from warm habitats, while there is no detectable difference in optimum and ceiling temperatures between habitats. Seed germination in response to water potential differed among the five studied populations. There was a negative correlation between the seed populations' base water potential for 50% (Ψ b(50)) germination and their hydrotime constant (θ H). The thermal time and hydrotime models were good predictors of five populations' germination time in response to temperature and water potentials. Consequently, future studies should consider the effects of maternal environmental conditions on seed germination when seeking effective strategies for controlling hemiparasitic weeds in alpine regions.

Effects of Stellera chamaejasme removal on the nutrient stoichiometry of S. chamaejasme-dominated grasslands in the Qinghai-Tibetan plateau.

BACKGROUND: Stoichiometric relations drive powerful constraints in several fundamental ecosystem processes. However, limited studies have been conducted on the ecological stoichiometry of plants after the change of community composition induced by Stellera chamaejasme removal in alpine grassland in the Qinghai-Tibetan Plateau. METHODS: We investigated the effects of S. chamaejasme removal on ecological stoichiometry by estimating the C:N:P stoichiometry in species, functional group and community levels of the ecosystem. The interactions between different species, functional groups and correlation with soil nutrient, responding to S. chamaejasme removal were also analyzed. RESULTS: For the plants that became dominant after S. chamaejasme removal (SR), N content decreased and their C:N increased. S. chamaejasme removal significantly affected the nutrient stoichiometry of different functional groups. Specifically, Gramineae in the SR sites had decreased N content and N:P, and increased C:N; however, forbs had increased N content, C:P and N:P and decreased P content and C:N. At the community level, N content was lower and C:N higher in SR communities compared to CK. The N content of the plant community was positively correlated with soil total N content. S. chamaejasme removal could change the nutrient balance from species level, to functional group level, and to community level. Thus, supplementary measures might be cooperated with S. chamaejasme removal for the recovery of S. chamaejasme-dominated degraded grassland. These results provide insight into the role of S. chamaejasme in ecological protection and conservation, and the conclusions from this study could be used to develop effective and sustainable measures for S. chamaejasme control in the Qinghai-Tibetan Plateau.

A toxic endophyte-infected grass helps reverse degradation and loss of biodiversity of over-grazed grasslands in northwest China.

Overgrazing of China's grasslands is increasingly causing biodiversity to decline. In degenerated grasslands of northwest China endophyte (Epichloё gansuensis) infected Achnatherum inebrians (drunken horse grass) is becoming widely distributed because of its toxicity to livestock. In this study, we investigated the ecological consequences of endophyte toxicity in this native grass, at three sites in northwest China, by comparing seed production of plant species and arthropod abundance in overgrazed grasslands with and without the presence of A. inebrians. Our findings demonstrate that the presence of endophyte infected A. inebrians reduces the loss of plant and arthropod biodiversity by providing a protected nursery free of animal grazing. Therefore, A. inebrians, typically regarded as an unwanted toxic invader by pastoralists, should be viewed as beneficial for grasslands as its presence maintains plant and arthropod biodiversity, and provides a foundation stone in the reconstruction and restoration of these grassland ecosystems.

Molecular Phylogeny of Grassland Caterpillars (Lepidoptera: Lymantriinae: Gynaephora) Endemic to the Qinghai-Tibetan Plateau.

Gynaephora (Lepidoptera Erebidae: Lymantriinae) is a small genus, consisting of 15 nominated species, of which eight species are endemic to the Qinghai-Tibetan Plateau (QTP). In this study, we employed both mitochondrial and nuclear loci to infer a molecular phylogeny for the eight QTP Gynaephora spp. We used the phylogeny to estimate divergence dates in a molecular dating analysis and to delimit species. This information allowed us to investigate associations between the diversification history of the eight QTP species and geological and climatic events. Phylogenetic analyses indicated that the eight QTP species formed a monophyletic group with strong supports in both Bayesian and maximum likelihood analyses. The low K2P genetic distances between the eight QTP species suggested that diversification occurred relatively quickly and recently. Out of the eight species, five species were highly supported as monophyletic, which were also recovered by species delimitation analyses. Samples of the remaining three species (G. aureata, G. rouergensis, and G. minora) mixed together, suggesting that further studies using extensive population sampling and comprehensive morphological approaches are necessary to clarify their species status. Divergence time estimation results demonstrated that the diversification and speciation of Gynaephora on the QTP began during the late Miocene/early Pliocene and was potentially affected by the QTP uplift and associated climate changes during this time.