Drug resistance and susceptibility testing of Gram negative bacterial isolates from healthy cattle with different β – Lactam resistance Phenotypes from Shandong province China

The objective of this study was to evaluate the effectiveness of common antibiotics against different microorganisms in apparently healthy cattle in Shandong province and its suburb. A total of 220 nasal swab samples were collected and cultured for bacteriological evaluation. All the bacteria isolates after preliminary identification were subjected to antibiogram studies following disc diffusion method. It was found in the study that E. coli is the most commonly associated isolate (21%), followed by Klebsiella spp. (18%), Pseudomonas aeruginosa (13%), Salmonella spp. (15%), Shigella spp (12%), and Proteus spp (11%). While the antibiogram studies reveled that highest number of bacterial isolates showed resistance to Ampicillin (95%), followed by Augmentin (91%), Cefuroxime (85%) and Tetracycline (95%) of (Escherichia coli and Klebsiella spp). In the case of pseudomonas spp. and Salmonella the highest resistance was showed by Ampicillin (90%) followed by Amoxicillin + Clavulanic Acid (80%), Cefixime (90%), and Erythromycin (80%). In Shigella spp and Salmonella spp highest resistance was showed by Amoxicillin, Ceftazidime, Augmentin (60%), and Amoxicillin + Clavulanic Acid (50%). It is concluded that in vitro antibiogram studies of bacterial isolates revealed higher resistance for Ampicillin, Augmentin, Cefuroxime, Cefixime, Tetracycline, Erythromycin, and Amoxicillin + Clavulanic Acid. The high multiple Antibiotics resistance indexes (MARI) observed in all the isolates in this study ranging from 0.6 to 0.9. MARI value of >0.2 is suggests multiple antibiotic resistant bacteria and indicate presence of highly resistant bacteria.

Control of Reactive Oxygen Species through Antioxidant Enzymes Plays a Pivotal Role during the Cultivation of Neopyropia yezoensis

Neopyropia yezoensis is an economically important marine crop that can survive dehydrating conditions when nets are lifted from seawater. During this process, production of oxygen radicals and the resulting up-regulation of antioxidant enzymes mediated by the abscisic acid (ABA) signaling pathway played an important role. However, there were no reports about the significance regarding the protection of seaweed throughout the entire production season. Especially, in new aquatic farms in Shandong Province that were formed when traditional N. yezoensis cultivation moved north. Here, we determined the levels of ABA, hydrogen peroxide (H2O2), soluble protein, chlorophyll, and cell wall polysaccharides in samples collected at different harvest periods from Jimo aquatic farm, Shandong Province. The activities and expression of NADPH oxidase (NOX) and antioxidant enzymes in the corresponding samples were also determined. Combined with the monitoring data of sea surface temperature and solar light intensity, we proposed that the cultivation of N. yezoensis in Shandong Province was not affected by high-temperature stress. However, photoinhibition in N. yezoensis usually occurs at noon, especially in March. Both the activities and the expression of NOX and antioxidant enzymes were up-regulated continuously. It is reasonable to speculate that the reactive oxygen species (ROS) produced by NOX induced the up-regulation of antioxidant enzymes through the ABA signaling pathway. Although antioxidant enzymes play a pivotal role during the cultivation of N. yezoensis, the production of ROS also caused a shift in gene expression, accumulation of secondary metabolites, and even decreased the chlorophyll pool size, which eventually led to a decrease in algae assimilation. Accordingly, we suggest that the dehydration of N. yezoensis nets should be adopted when necessary and the extent of dehydration should be paid special consideration to avoid an excessive cellular response caused by ROS.

First Report of New Bacterial Leaf streak of Rice Caused by Pantoea ananatis in China

Rice (Oryza sativa L.) is the largest grain crop, accounting for about 40 % of the total grain production in China. In mid-July 2021, bacterial leaf streak-like disease emerged in rice varieties Chunyou584 and Yongyou2604 in Linyi city, Shandong Province, China. Disease incidences of the disease ranged from 80% to 90% in the surveyed fields. Infected rice leaves displayed dark green to yellowish-brown water-soaked thin streaks, and a large amount of beaded yellow oozes were observed on the lesions. After drying, there were gelatinous granules that were not easy to fall off and spread between leaf veins (Fig.S1A). According to the field symptoms of this disease, it was preliminarily suspected to be rice bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc), which is a guaranteed disease in China. To isolate the causal agent, leaf discs (~1 cm2) of diseased leaves were collected from the margins of the lesions, surface sterilized and ground into pieces in sterile double distilled water. The 10-3, 10-4 and 10-5 dilutions were spread onto peptone sugar agar (PSA) and incubated at 28°C for 36 hours. Yellow mucous bacterial colonies were consistently obtained on PSA medium. To identify the pathogen, fragments of the 16S rDNA, leuS and rpoB were amplified and sequenced using the primers previously reported (Yu et al. 2021). Three strains (LY01, LY02 and LY03) showed identical colony morphology and LY01 was used for further analyses. Sequence analyses showed that the fragments of 16S rDNA (955 bp, GenBank accession number: OK261898), leuS (755 bp, GenBank accession number: OK298387) and rpoB (926 bp, GenBank accession number: OK298388) of strain LY01 shared 99.16%, 99.46% and 100% similarities with those of Pantoea ananatis TZ39 (GenBank accession numbers: CP081342.1 for 16S rDNA, MW981338.1 for leuS and MW981344.1 for rpoB), respectively, which suggest the pathogenic bacterial strain LY01 isolated is P. ananatis. In addition, the single colony of P. ananatis LY01 was shown as Fig. S2B. Furthermore, pathogenicity tests were also performed according to the following steps. Bacterial suspension at OD600=0.1 was inoculated into eight rice leaves of four healthy rice plants (Chunyou 584) at 25-33°C and 60%-80% relative humidity in the field using a clipping method (Yang et al. 2020) or spraying methods, and sterile distilled water was as negative control. The clipped leaves (Fig. S1B) and spray-inoculated leaves (Fig. S1C) showed dark green water-soaked streaks at 14 days after inoculation, respectively, which showed similar symptoms with those samples collected from the fields (Fig. S1A). On contrary, the control rice leaves remained healthy and symptomless (Fig. S2A). The bacterium was re-isolated in the inoculated rice leaves and the re-isolated bacterial isolates, which was confirmed by sequencing 16S rDNA, leuS and rpoB, incited the same symptoms as in fields, which fulfills Koch’s postulates. In the past decade, P. ananatis was reported to result in grain discoloration and leaf blight in China (Yan et al. 2010; Xue et al. 2020, Yu et al. 2021), which could result in 40% - 60% yield losses. To our best knowledge, this is the first report of the bacterial leaf streak-likely disease occurred in Shandong Province caused by P. ananatis, so we named it as Pantoea leaf streak of rice. Although P. ananatis was also reported in Zhejiang province and Jiangxi province, which caused leaf streak lesions on rice, the disease symptoms are completely different from those of Pantoea leaf streak of rice. To the best of our knowledge, this is the first report of Pantoea leaf streak of rice caused by P. ananatis. This study provides sloid evidence that Pantoea leaf streak of rice in Eastern China can be caused by the new pathogen, P. ananatis, rather than Xoc as traditionally assumed. Disease development and quarantine of the new Pantoea leaf streak of rice disease caused by P. ananatis on rice need more attention in the near future.

First Report of Colletotrichum truncatum Causing Anthracnose on Oxalis corniculata in China

Oxalis corniculata L., which belongs to the family Oxalidaceae R. Br., is a very common perennial herb. It is usually planted on bare land or under the forest as landscaping plants, and the whole plant can be used for its medicinal values of clearing heat, detoxification and detumescence. In August 2019, typical symptoms of anthracnose on O. corniculata leaves were observed in the green belt on the campus of Shandong University of Technology (36.81°N, 117.99°E), Shandong Province, China. The disease incidence was above 40% by investigating more than 300 m2 of planting area. Most of O. corniculata are planted under the forest where the disease is found, mainly in the environment with high relative humidity and less ventilation. The infected leaves appeared initially as tawny oval or irregular spots, and then the lesions enlarged gradually until the leaves became dieback or wholly withered, which greatly reduced the landscape effect of O. corniculata. Diseased leaves were collected by cutting into small pieces and sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite (NaClO) for 60 s, rinsed with sterile deionized water for three times. Each air-dried tissue segment was cultured on potato dextrose agar (PDA) and incubated at 25℃ for 5 to 7 days in the dark (Zhu et al. 2013). Fifteen isolates were obtained from 20 symptomatic leaves and the cultures were initially gray white, subsequently became grayish to dark green after 7 days, with copious gray aerial mycelium and black microsclerotia. Three isolates were verified by the amplification of DNA sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), histone H3 (H3) and chitin synthase (CHS1) genes, using the primer pairs GDF1/GDR1, ACT-512F/ACT-783R, CYLH3F/CYLH3R, and CHS-79F/CHS-234R (Damn et al. 2019, Fu et al. 2019, Liu et al. 2013), respectively. The sequenced genes (GenBank accession no. OK017473, OK159078, OK159076, OK159077) shared 99.62 to 100.00% nucleotide identity with the corresponding genes of Colletotrichum truncatum strain UASB-Cc-10 (GenBank accession no. KF322064.1, KF322055.1, KF322073.1, KF319059.1), respectively, which was consistent with the morphological identification (Sawant et al. 2012). Pathogenicity test was performed with six healthy O. corniculata plants infected with mycelial plugs (about 3 mm in diameter) of three C. truncatum isolates from a 5-day-old culture, while the negative controls on the same leaves were inoculated with sterile PDA plugs. All plants were placed in a greenhouse at 25 to 30℃ with 90% relative humidity. The experiment was conducted three times. Five days later, all inoculated leaves appeared brown sunken spots, whereas no symptoms appeared on negative controls. The same pathogens, C. truncatum, were identified from the inoculated leaves on the basis of morphological and molecular characteristics as described above, confirming Koch’s postulates. To our knowledge, anthracnose caused by C. truncatum on O. corniculata is the first report in China. The discovery of this new disease is beneficial to the application and protection of O. corniculata, a popular landscape and medicinal plant. References: Damn, U., et al. 2019. Stud. Mycol. 92:1. https://doi.org/10.1016/j.simyco.2018.04.001 Fu, M., et al. 2019. Persoonia 42:1. https://doi.org/10.3767/persoonia.2019.42.01 Liu, F., et al. 2013. Mycologia 105:844. https://doi.org/10.3852/12-315 Sawant, I. S., et al. 2012. New Dis. Rep. 25:2. https://doi.org/10.5197/j.2044-0588.2012.025.002 Zhu, L., et al. 2013. J. Phytopathol. 161:59. https://doi.org/10.1111/jph.12019 The author(s) declare no conflict of interest. Acknowledgments: This research was financially supported by the Top Talents Program for One Case One Discussion of Shandong Province and Academy of Ecological Unmanned Farm (2019ZBXC200).

The co-evolution of seaports and dry ports in Shandong province in China under the Belt and Road Initiative

Nowadays, Belt and Road Initiative (BRI) has been prosperous for a long time and brings plenty of opportunities to every aspect of China. As a coastal province with lots of port resources, Shandong province has implemented port integration, and there are some operation problems arising, too. In order to make the seaports and dry port reach their full potential, it is very crucial to explore the evolution trend of the ports, which can offer more chances for future development. Based on related theory, this article analyses the evolution of seaports and dry ports in Shandong Province, combined with the background of BRI. After showing the trend of port evolution, this paper points out the co-evolution mechanism of seaports and dry ports and describe a dry port-seaport logistics network in Shandong Province, which results from the mutual influence of seaports and dry ports. Finally, considering the influence of the BRI, this article points out that Shandong Province should take the inland cities as the basis and consider the various costs of the ports to fully take part in the Belt and Road Initiative, when constructing a logistics network.