Effects of Grazing and Leaf Spot Disease on the Structure and Diversity of Phyllosphere Microbiome Communities in Leymus chinensis.

Leymus chinensis is a high-quality forage with wide distribution. Disease is an important factor affecting the yield and quality of L. chinensis. To investigate the effect of grazing on the phyllosphere microbiome community and leaf spot disease in L. chinensis, high-throughput sequencing technology was used to study the differences in the composition and structure of the phyllosphere fungal and bacterial communities of healthy and diseased leaves under different grazing intensities. The results showed that grazing significantly reduced leaf spot disease incidence and severity. There were significant differences in the phyllosphere microbiome composition between healthy and diseased leaves, and interestingly, diseased leaves showed more complex microbial activity. Grazing altered the relative abundance of micro-organisms and affected microbial dispersal and colonization either directly through behavior or indirectly by altering plant community structure. In this study, we found that the phyllosphere microbiome responded strongly to pathogen infection, and that plants recruited beneficial microbes to protect themselves after disease development. Grazing could regulate microbial community composition and structure, either directly or indirectly, and plays a crucial role in maintaining the health of L. chinensis.

First Report of Bipolaris zeae Causing Leaf Spot on Setaria viridis in China.

Green bristlegrass (Setaria viridis) is an annual dominant herb in arid sandy grasslands with strong drought resistance to water stress (Valença et al. 2020). In August 2021, brown or dark tan spots on the leaves of S. viridis were observed on natural grassland in Qingyang city, Gansu Province, China (36.55°N, 107.32°E). Leaf disease incidence was around 13%, and the infected area of each leaf was about 20%. For isolation, 30 tissue pieces (5×5 mm) from 10 symptomatic leaf samples were surface-sterilized with 70% ethanol for 30 seconds and rinsed three times with steriled distilled water. Then these tissues were placed on the potato dextrose agar (PDA) at 25 °C, and incubated in dark from 2 to 5 days. Sixteen single-spored cultures with consistent colony characteristics were obtained by single spore isolation as described in Manamgoda et al. (2012). Colonies on PDA were irregular shapes with black color and white edge; conidia were dark brown with septate, cylindrical, straight or flexuous geniculate at the upper end, 22.7 to 69.38×9.2 to 17.6 µm (n = 50; average size 40.8×13.2 µm) under microscope (40X). The internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GPD) regions of the representative isolate 83HXGWC, 84HXGWC and 85HXGWC were amplified using the primers of ITS1/ITS4 and GPD1/GPD2 (Manamgoda et al. 2012). All the amplified gene sequences were deposited into GenBank with accession numbers ITS (OP703331, OQ978850, and OQ978851) and GPD (OQ559683, OQ992505, and OQ992506). BLAST analysis of both the two segments showed 100% identity with those reported sequences of ex-type isolate BRIP 11512 (KJ415538 and KJ415408) of B. zeae Sivan (Tan et al. 2014; Bhunjun et al. 2020), respectively. In the maximum-likelihood phylogenetic tree, 83-HXGWC, 84HXGWC and 85HXGWC strains were grouped with the reference sequences of B. zeae with a high bootstrap supporting values of 100%. Five healthy green bristlegrass (6 weeks) plants in pots were sprayed with conidial suspensions (1×106 conidia ml-1). Another five pots were sprayed with sterilized distilled water (controls). Then all pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse maintained at 18 to 25 °C. After incubation for 14 days, the typical symptoms of leaf spots developed from brown to dark brown on the plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The measurement of pathogenicity was carried out three times. The same pathogens were consistently reisolated from inoculated leaves, and was confirmed as B. zeae based on morphological and molecular analyses, fulfilling Koch's postulates. This study provides a new record of B. zeae on green bristlegrass on natural grassland in China, which can potentially impose disease challenges to other crops. Since, B. oryzae has been known to cause disease in cereal crops (Manamgoda et al. 2014), this can be a potential alternate source of survival of this pathogen.

Co-infection by Soil-Borne Fungal Pathogens Alters Disease Responses Among Diverse Alfalfa Varieties.

Fusarium oxysporum f. sp. medicaginis (Fom) and Rhizoctonia solani (Rs) are the major soil-borne fungal pathogens that pose severe threats to commercial alfalfa production in China. However, the effects of Fom and Rs co-infection on alfalfa and whether co-infection alters disease resistance responses among diverse varieties remain unknown. A collection of 80 alfalfa varieties (Medicago sativa) originated from seven countries were used to study the effects of Fom and Rs co-infection on alfalfa and host resistance responses. The co-infection resulted in more severe disease and reductions in growth and biomass allocation across varieties in comparison with either single infection by Fom or Rs; in addition, root morphology was much more strongly altered by the co-infection. Principal component analysis based on all plant traits showed that varieties under the co-infection were related to the single infection by Rs, being separated from Fom, and hierarchical clustering found differential response patterns among varieties upon co-infection compared with either single infection, with most varieties being highly susceptible to the co-infection. Furthermore, varieties that were most resistant to either single infection were not effective to co-infection, and there was no individual variety with resistance to both pathogens singly and co-infected. This study reveals for the first time that the co-infection by Fom and Rs alters disease resistance responses among diverse alfalfa varieties and provides useful information for developing alfalfa varieties with resistance to the co-occurrence of different soil-borne pathogens.

[In vitro study on bFGF expression of rat astrocytes after mechanical injury].

OBJECTIVE: To study the alteration of basic fibroblast growth factor (bFGB) expression in astrocytes in vitro after mechanical injury and to understand the repair mechanism of brain injury. METHODS: Astrocytes were isolated from cerebral cortex of SD rats born in 24 hours, and then cultured and purified. The cultured astrocytes were randomly divided into control group and injury groups that were subjected to mechanical injury at 30 min, 1h, 3h, 6h, 12h, 24h, 3d, and 7d. The levels of bFGF expression in the astrocytes after injury were detected by ABC immunohistochemistry. RESULTS: More than 95% of the cultured cells were astrocytes. The levels of bFGF expression werevery low in the control group. On the other hand, increased levels of bFGF expression could be observed at 1-3h after injury. The expression levels increased significantly at 6-12h, reached peak level at 24h, remained at the high level up to 3 days, and the decreased gradually. CONCLUSION: The changes of bFGF expression levels in cultured astrocytes in vitro after mechanical injury are similar to that observed in vivo experimental model, both of which show time-dependant characteristic, with only slightly earlier expression of bFGF observed in vitro. Thus, the expression of bFGF after injury can be one of evidences for estimation of brain injury intervals. the cell injury model in vitro may have superiority in the study of the molecule mechanism of tissue and cell injury.

[Changes of HSP70, bFGF and TGF-beta1 expression in rat brain after concussion].

OBJECTIVE: To study the changes of expression of relevant factors in rat brain after concussion injury and to provide scientific basis for forensic estimation of brain injury interval. METHODS: Brain tissues were sampled from the established SD rat animal model of brain concussion, routinely processed and stained with HE and immunohistochemically stained with antibodies directed against heat shock protein 70 (HSP70), transforming growth factor beta 1 (TGF-beta1) and basic fibroblast growth factor (bFGF). The sections were examined under light microscope with IMAGE analytical system and homologous statistical analysis. RESULTS: The expression of HSP 70 was observed in 30 minutes after brain injury. The amount of neurons expressing HSP 70 increased gradually, reached its peak at 12 hours and then declined at 24 hours after brain injury. The expression of bFGF was observed 3 hours after injury in brain stem, reached its peak at 12 hours, and then declined. The expression of TGF-beta1 was detected 6-24 hours after brain injury, remained at its peak up to 3 days. CONCLUSION: Brain injury can induce a chronological expression of HSP70, bFGF and TGF-beta1. The results can be a potential for estimating the age of brain injury using several markers.