Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

Genetic diversity and molecular characterization of Cucumber mosaic cucumovirus (CMV) subgroup II infecting Spinach (Spinacia oleracea) and Pea (Pisum sativum) in Pothwar region of Pakistan

Cucumber mosaic virus (CMV) is a tremendous threat to vegetables across the globe, including in Pakistan. The present work was conducted to investigate the genetic variability of CMV isolates infecting pea and spinach vegetables in the Pothwar region of Pakistan. Serological-based surveys during 2016-2017 revealed 31.70% overall CMV disease incidence from pea and spinach crops. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) revealed that all the positive isolates belong to CMV subgroup II. Two selected cDNA from ELISA-positive samples representing each pea and spinach crops were PCR-amplified (ca.1100 bp) and sequenced corresponding to the CMV CP gene which shared 93.7% nucleotide identity with each other. Both the sequences of CMV pea (AAHAP) and spinach (AARS) isolates from Pakistan were submitted to GenBank as accession nos. MH119071 and MH119073, respectively. BLAST analysis revealed 93.4% sequence identity of AAHAP isolate with SpK (KC763473) from Iran while AARS isolate shared maximum identity (94.5%) with the strain 241 (AJ585519) from Australia and clustered with some reference isolates of CMV subgroup II from UK (Z12818) and USA (AF127976) in a Neighbour-joining phylogenetic reconstruction. A total of 59 polymorphic (segregating) sites (S) with nucleotide diversity (π) of 0.06218 was evident while no INDEL event was observed in Pakistani isolates. The evolutionary distance of Pakistani CMV isolates was recorded as 0.0657 with each other and 0.0574-0.2964 with other CMV isolates reported elsewhere in the world. A frequent gene flow (Fst = 0.30478 <0.33) was observed between Pakistani and earlier reported CMV isolates. In genetic differentiation analysis, the value of three permutation-based statistical tests viz; Z (84.3011), Snn (0.82456), and Ks* (4.04042) were non-significant. The statistical analysis revealed the values 2.02535, 0.01468, and 0.71862 of Tajima's D, Fu, & Li’s F* and D* respectively, demonstrating that the CMV population is under balancing selection.

First Report of Rhizome Rot Caused by Pectobacterium brasiliense on Ginger in China

In August 2020, ginger (Zingiber officinale) rhizomes (cv. Mianjiang) showing soft rot symptoms were observed in a field in Tayang Village, Fengrun District, Tangshan, Hebei Province (North China). The disease incidence in that field (15 ha in size) was more than 20%. Symptomatic rhizomes (brown and water-soaked) were surface-sterilized in 75% ethanol for 60 sec and then three successive rinses with sterile distilled water. Rhizomes were cut into pieces ca. 0.5 cm in length, and then were soaked in 500 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the lysogeny broth (LB) medium. And LB plates were incubated at 28°C for 24 h. Five single colonies were picked from each LB plate and restreaked three times for purity. Endophytic bacteria were also isolated from asymptomatic rhizomes as control. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R (Weisburg et al. 1991) and sequenced. The results of BLASTN against NCBI nr of the 16S rDNA amplicons suggested that the most isolates (8/10) obtained from the rotten rhizomes belonged to the genus Pectobacterium, and few isolates (2/10) were Enterobacter spp.. Only Enterobacter spp. were isolated from asymptomatic rhizomes. Since all Pectobacterium isolates showed identical 16S rDNA sequence, thus, only two isolates were selected for further analysis. Pectobacterium isolates TS20HJ1 and TS20HJ2 (MZ853520, MZ853521) represent isolates from two plant individuals. To determine the species of the rhizome rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with five housekeeping genes acnA, icdA, mdh, proA and rpoS (MZ994717-MZ994726) (Ma et al. 2007; Waleron et al. 2008), and a phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between the two isolates. The result of phylogenetic analysis showed that the ginger rhizome isolates clustered with P. brasiliense type strain IBSBF1692T (Duarte et al. 2004; Nabhan et al. 2012). Ginger seedlings (cv. Mianjiang) were inoculated with the isolate TS20HJ1 by injecting 10 µl of bacterial suspensions (108 CFU·mL-1) into the rhizomes, or injected with 10 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Ten days after inoculation, only the bacteria-inoculated rhizomes showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected rhizomes and were identified with MLSA gene sequencing, fulfilling Koch’s postulates. P. brasiliense causes soft rot of a wide range of economically important crops (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing rhizome rot of ginger in China. The rhizome rot caused 20-25% yield loss on average in Tangshan region in 2020, which poses a significant threat to the local ginger farming. Further research on epidemiology and disease management options is needed.

Longitudinal Impact of WTC Dust Inhalation on Rat Cardiac Tissue Transcriptomic Profiles

First responders (FR) exposed to the World Trade Center (WTC) Ground Zero air over the first week after the 9/11 disaster have an increased heart disease incidence compared to unexposed FR and the general population. To test if WTC dusts were causative agents, rats were exposed to WTC dusts (under isoflurane [ISO] anesthesia) 2 h/day on 2 consecutive days; controls received air/ISO or air only. Hearts were collected 1, 30, 240, and 360 d post-exposure, left ventricle total RNA was extracted, and transcription profiles were obtained. The data showed that differentially expressed genes (DEG) for WTC vs. ISO rats did not reach any significance with a false discovery rate (FDR) < 0.05 at days 1, 30, and 240, indicating that the dusts did not impart effects beyond any from ISO. However, at day 360, 14 DEG with a low FDR were identified, reflecting potential long-term effects from WTC dust alone, and the majority of these DEG have been implicated as having an impact on heart functions. Furthermore, the functional gene set enrichment analysis (GSEA) data at day 360 showed that WTC dust could potentially impact the myocardial energy metabolism via PPAR signaling and heart valve development. This is the first study showing that WTC dust could significantly affect some genes that are associated with the heart/CV system, in the long term. Even > 20 years after the 9/11 disaster, this has potentially important implications for those FR exposed repeatedly at Ground Zero over the first week after the buildings collapsed.

Spatiotemporal variations in exposure: Chagas disease in Colombia as a case study

Age-stratified serosurvey data are often used to understand spatiotemporal trends in disease incidence and exposure through estimating the Force-of-Infection (FoI). Typically, median or mean FoI estimates are used as the response variable in predictive models, often overlooking the uncertainty in estimated FoI values when fitting models and evaluating their predictive ability. To assess how this uncertainty impact predictions, we compared three approaches with three levels of uncertainty integration. We propose a performance indicator to assess how predictions reflect initial uncertainty.In Colombia, 76 serosurveys (1980–2014) conducted at municipality level provided age-stratified Chagas disease prevalence data. The yearly FoI was estimated at the serosurvey level using a time-varying catalytic model. Environmental, demographic and entomological predictors were used to fit and predict the FoI at municipality level from 1980 to 2010 across Colombia.A stratified bootstrap method was used to fit the models without temporal autocorrelation at the serosurvey level. The predictive ability of each model was evaluated to select the best-fit models within urban, rural and (Amerindian) indigenous settings. Model averaging, with the 10 best-fit models identified, was used to generate predictions.Our analysis shows a risk of overconfidence in model predictions when median estimates of FoI alone are used to fit and evaluate models, failing to account for uncertainty in FoI estimates. Our proposed methodology fully propagates uncertainty in the estimated FoI onto the generated predictions, providing realistic assessments of both central tendency and current uncertainty surrounding exposure to Chagas disease.

Biocontrol Potential of Chitin and Chitosan Extracted from Black Soldier Fly Pupal Exuviae against Bacterial Wilt of Tomato

Globally, Ralstonia solanacearum (Smith) is ranked one of the most destructive bacterial pathogens inducing rapid and fatal wilting symptoms on tomatoes. Yield losses on tomatoes vary from 0 to 91% and most control measures are unaffordable to resource-poor farmers. This study investigated the antimicrobial activities of chitin and chitosan extracted from black soldier fly (BSF) pupal exuviae against R. solanacearum. Morphological, biochemical, and molecular techniques were used to isolate and characterize R. solanacearum for in vitro pathogenicity test using disc diffusion technique. Our results revealed that BSF chitosan significantly inhibited the growth of R. solanacearum when compared to treatments without chitosan. However, there was no significant difference in the antibacterial activities between BSF and commercial chitosan against R. solanacearum. Soil amended with BSF-chitin and chitosan demonstrated a reduction in bacterial wilt disease incidence by 30.31% and 34.95%, respectively. Whereas, disease severity was reduced by 22.57% and 23.66%, when inoculated tomato plants were subjected to soil amended with BSF chitin and chitosan, respectively. These findings have demonstrated that BSF pupal shells are an attractive renewable raw material for the recovery of valuable products (chitin and chitosan) with promising ability as a new type of eco-friendly control measure against bacterial wilt caused by R. solanacearum. Further studies should explore integrated pest management options that integrate multiple components including insect-based chitin and chitosan to manage bacterial wilt diseases, contributing significantly to increased tomato production worldwide.

The global epidemiology of chikungunya from 1999 to 2020: A systematic literature review to inform the development and introduction of vaccines

Chikungunya fever is an acute febrile illness that is often associated with severe polyarthralgia in humans. The disease is caused by chikungunya virus (CHIKV), a mosquito-borne alphavirus. Since its reemergence in 2004, the virus has spread throughout the tropical world and several subtropical areas affecting millions of people to become a global public health issue. Given the significant disease burden, there is a need for medical countermeasures and several vaccine candidates are in clinical development. To characterize the global epidemiology of chikungunya and inform vaccine development, we undertook a systematic literature review in MEDLINE and additional public domain sources published up to June 13, 2020 and assessed epidemiological trends from 1999 to 2020. Observational studies addressing CHIKV epidemiology were included and studies not reporting primary data were excluded. Only descriptive analyses were conducted. Of 3,883 relevant sources identified, 371 were eligible for inclusion. 46% of the included studies were published after 2016. Ninety-seven outbreak reports from 45 countries and 50 seroprevalence studies from 31 countries were retrieved, including from Africa, Asia, Oceania, the Americas, and Europe. Several countries reported multiple outbreaks, but these were sporadic and unpredictable. Substantial gaps in epidemiological knowledge were identified, specifically granular data on disease incidence and age-specific infection rates. The retrieved studies revealed a diversity of methodologies and study designs, reflecting a lack of standardized procedures used to characterize this disease. Nevertheless, available epidemiological data emphasized the challenges to conduct vaccine efficacy trials due to disease unpredictability. A better understanding of chikungunya disease dynamics with appropriate granularity and better insights into the duration of long-term population immunity is critical to assist in the planning and success of vaccine development efforts pre and post licensure.

First Report of Paraphoma chrysanthemicola Causing Crown and Leaf Rot of Atractylodes lancea in China

Atractylodes lancea is an important traditional Chinese medicinal plant whose rhizome is used for treating complaints such as rheumatic diseases, digestive disorders, night blindness and influenza. Jiangsu Province is the optimal cultivation location for high-quality A. lancea rhizome. Since June 2019, symptoms of crown rot and leaf rot were observed in about 10-20% of the A. lancea in a plantation (31° 36' 1" N, 119° 6' 40" W) in Lishui, Jiangsu, China. Lesions occurred on the stem near the soil line and on the leaves (Fig. 1A). Disease incidence reached approximately 80-90% by September, 2021 (Fig. 1B) and resulted in severe loss of rhizome and seed yields. For pathogen isolation, ten samples of symptomatic stem segments and ten diseased leaves were collected, surface-sterilized using 5% NaClO solution, rinsed with sterile water, cut into 0.5-2 cm segments, and plated to potato dextrose agar (PDA), and then incubated at 30°C in darkness. Pure cultures of four isolates showing morphological characteristics of Paraphoma spp. were obtained, identified as a single P. chrysanthemicola strain, and named LSL3f2. Newly formed colonies initially consisted of white mycelia; the five-day-old colonies developed a layer of whitish grey mycelia with a grey underside. 20-day-old colonies had white mycelium along the margin and with a faint yellow inner circular part with irregular radial furrows, and the reverse side looking caramel and russet (Fig. 1C). Pycnidia were subglobose (diameter: 5 to 15 μm; Fig. 1D). Unicellular, bicellular or strings of globose or subglobose chlamydospores developed from hyphal cells (Fig. 1E and 1F). The internal transcribed spacer (ITS) region and large subulin-28S of LSL3f2 were cloned using primers ITS1/ITS4 and LR0R/LR7 (Aveskamp et al. 2010, Li et al. 2013), and deposited in GenBank (OK559658 and OK598973, respectively). BLASTn search and phylogenetic analysis showed the highest identity between LSL3f2 and P. chrysanthemicola sequences (Fig. 1G) and confirmed LSL3f2 as P. chrysanthemicola. Koch’s postulates were completed using one-month-old vegetatively propagated A. lancea plantlets growing on autoclaved vermiculite/peat mixture at 26°C with a light/dark cycle of 12/12 hours. Each plantlet was inoculated with 5 ml of conidial suspension in water (1 × 108 cfu/ml) by applying to soil close to the plantlet, with sterile water used as a mock control (n = 10). By 20 days post-inoculation, inoculated plantlets showed a range of disease symptoms consistent to those observed in infested fields (Fig. 1H). Pathogenicity was additionally confirmed using detached leaves inoculated with a colonized agar plug of LSL3f2 or an uninoculated control comparison (diameter = 5 mm) and incubated at 26℃ in the dark. Five to seven days post-inoculation, detached leaves showed leaf rot symptoms including lesions, yellowing and withering consistent with those in infested fields, while control leaves remained healthy (n = 10, Fig. 1I). The pathogen was reisolated from the diseased plantlets and detached leaves, in both cases demonstrating the micromorphological characteristics of LSL3f2. P. chrysanthemicola has been reported to cause leaf and crown rot on other plants such as Tanacetum cinerariifolium (Moslemi et al. 2018), and leaf spot on A. japonicain (Ge et al. 2016). However, this is the first report of P. chrysanthemicola causing crown and leaf rot on A. lancea in China.

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).