Eocene tortoise beetles from the Green River Formation in Colorado,U.S.A. (Coleoptera: Chrysomelidae: Cassidinae)

Abstract The fossil history of leaf beetles (Chrysomelidae) is relatively poorly documented despite an abundance of available material. Of particular interest is the origin and radiation of the diverse tortoise beetles, a derived group within Cassidinae s.l. (=Cassidinae + Hispinae) defined by the exophagous life history and specialized morphology of the immature stages. Cassidinae is also a group with relatively few fossil records that can be assigned with any degree of certainty. Here we report two of the oldest definitive tortoise beetle fossils, Eosacantha delocranioides gen.n. et sp.n. and Denaeaspis chelonopsis gen.n. et sp.n. , from the Eocene Green River Formation (ca. 47 million years old) in northwestern Colorado, U.S.A. Owing to the fine level of preservation, many important features can be observed and are coded into the recent cladistic analysis for the subfamily. Phylogenetic analysis highlights that both genera have affinities with modern lineages, one restricted to the Old World and the other restricted to the Neotropics. Although Cassidinae as a whole extend into the Cretaceous, the available information suggests that the tortoise beetles perhaps originated and diversified during the Early Tertiary. As such, the morphological and biological transitions from the leaf‐mining hispiforms to the distinctive tortoise‐like cassidiforms, with their elaborate defensive larval shields and other unique behaviours, probably took place during the latest Paleocene or earliest Eocene. These Green River fossils are the oldest yet to document the specialized morphology associated with the transition in cassidine feeding and immature biology.     

Determining Risk Factors for Dengue Fever Severity in Jeddah City,a Case-Control Study (2017)

Dengue fever is a major public health problem in Saudi Arabia. Unfortunately, preventive strategies are still deficient. It can progress to severe and lethal forms, and available knowledge does not allow early prediction of which cases of dengue fever (DF) will progress to dengue hemorrhagic fever (DHF). The aim of this study was to evaluate the host and viral factors that could play a role in the progression of severe dengue cases in the frame of the revised 2009 WHO classification. Data were compiled from the Jeddah Dengue Fever Operation Room (DFOR) in the Maden Al-Fahd primary healthcare center in Jeddah. An unmatched case-control study was conducted on 123 severe cases, and 245 controls (non-severe cases) diagnosed during 2014–2016. Risk factors for severe dengue fever were secondary infection (p = 0.02), and co-morbidities, particularly diabetes and hypertension (p < 0.001). Age, gender, nationality, socioeconomic status, viral serotype, and access to health care were not significantly associated with severe disease. The main risk factors for severe dengue fever were secondary infection, and co-morbidities (hypertension and diabetes). We recommend disseminating these data to stakeholders to improve dengue control interventions in periods with anticipated high incidence.Key words: Dengue fever, viral infection, case control, risk factors     

Intraspecies Variability in the Esterases and Acid Phosphatases of Four Species of the Paramecium aurelia Complex1

ABSTRACT. One hundred eighty-eight stocks of Paramecium primaurelia. P. biaurelia, P. tetraurelia. and P. octaurelia were grown axenically and screened for variation in four different esterases and acid phosphatase using starch gel electrophoresis. Major observations: frequency of intraspecies variation for these enzymes is much lower in these four species than in other organisms; hypervariability for two esterases occurs in P. biaurelia both in isolates from worldwide locales and in a restricted locale; clustering of variations occurs in a high proportion of variant stocks in all four species; frequency of intraspecies variation is highest in Central and South America for all four species; and geographical differentiation is lacking between stocks in the same species both for common as well as variant phenotypes despite the cosmopolitan distribution of these species. These results are not correlated with adaptations that favor inbreeding over outbreeding. nor is the possession of bacterial endosymbionts strongly correlated with enzyme variation. When the riequency of intraspecies variation was examined for the aurelia complex of species as a whole for 13 enzymes, mitochondrial DNA, and ribosomal DNA, differences between enzymes in frequency of variation could be seen, ranging from less than 2% for seven enzymes to 12.4% for glucosephosphate isomerase, a value similar to that observed for malic dehydrogenase, mitochondrial DNA, and ribosomal DNA in P. tetraurelia. The percentage of polymorphic enzyme loci in the complex as a whole was found to be much lower than that observed for other organisms. For the species more intensely studied in this paper the level of genetic polymorphism was also much lower, although P. biaurelia showed greater variability for two of the enzymes.     

In vitro Antagonistic Activity of Endophytic Fungi Isolated from Shirazi Thyme (Zataria multiflora Boiss.) against Monosporascus cannonballus

Endophytic fungi viz., Nigrospora sphaerica (E1 and E6), Subramaniula cristata (E7), and Polycephalomyces sinensis (E8 and E10) were isolated from the medicinal plant, Shirazi thyme (Zataria multiflora). In in vitro tests, these endophytes inhibited the mycelial growth of Monosporascus cannonballus, a plant pathogenic fungus. Morphological abnormalities in the hyphae of M. cannonballus at the edge of the inhibition zone in dual cultures with N. sphaerica were observed. The culture filtrates of these endophytes caused leakage of electrolytes from the mycelium of M. cannonballus. To our knowledge, this is the first report on the isolation and characterization of fungal endophytes from Z. multiflora as well as their antifungal effect on M. cannonballus.Key words: Zataria multiflora, antifungal, endophytic fungi, Monosporascus cannonballus

The term “Endophytes” denotes microorganisms that colonize plants’ internal tissues for part of or throughout their life cycle without producing any apparent adverse effect. The endophytic microorganisms include fungi, bacteria, and actinobacteria (Bacon and White 2000). Among them, fungi are the most common endophytic microorganisms (Staniek et al. 2008). Endophytic fungi are ecologically distinct polyphyletic groups of microorganisms, mostly belonging to the Ascomycota phylum (Jia et al. 2016). Several fungal endophytes have been shown to act as biological control agents for managing soil-borne plant pathogens (Toghueo et al. 2016).Zataria multiflora Boiss. (Synonyms: Zataria bracteata Boiss.; Zataria multiflora var. elatior Boiss), belonging to the Lamiaceae family is a traditional medicinal plant commonly used as a flavor ingredient in different types of foods (Sajed et al. 2013). Several medicinal properties of Z. multiflora, including antiseptic, anesthetic, antispasmodic, antioxidant, antibacterial, and immunomodulatory activities, have been documented (Sajed et al. 2013). However, studies on the endophytic microorganisms inhabiting Z. multiflora are limited (Mohammadi et al. 2016).Monosporascus cannonballus Pollack & Uecker (Ascomycota, Sordariomycetes, Diatrypaceae) is one of the most important phytopathogenic fungi causing root rot and vine decline disease in muskmelon. It causes sudden wilt and collapse of the plant at the fruiting stage, which may result in total yield loss (Martyn and Miller 1996). The fungus also infects pumpkin, cucumber, courgette, and watermelon plants (Mertely et al. 1993). The control of M. cannonballus in melon and other cucurbit crops is difficult because of the pathogen’s soil-borne nature. Earlier reports indicated that arbuscular mycorrhizal fungi (AMF) (Aleandri et al. 2015), hypovirulent isolates of M. cannonballus (Batten et al. 2000), Trichoderma spp. (Zhang et al. 1999), and antagonistic rhizobacteria (Al-Daghari et al. 2020) are effective agents for the reduction of M. cannonballus-induced root rot and vine decline of melon. In addition, it is well established that many endophytic fungi isolated from medicinal plants possess antimicrobial activity against phytopathogenic fungi (Jia et al. 2016). The objective of this study was to investigate the presence of endophytic fungi in Z. multiflora and to study theirs in vitro antagonistic activity against M. cannonballus.Z. multiflora plants (accession number 201100114) were obtained from Oman Botanic Garden, Al-Khoud, Sultanate of Oman. The plants were healthy, showing no apparent symptoms of any disease or pest infestation. A virulent isolate of M. cannonballus (ID14367), obtained from the roots of a melon plant showing root rot and vine decline (Al-Rawahi et al. 2018) was used in this study. The culture was maintained on potato dextrose agar (PDA) medium (Oxoid Ltd., Basingstoke, UK).To isolate endophytic fungi, Z. multiflora plants were washed in tap water to remove adhering soil particles. The leaves were separated, cut into small pieces, and surface-sterilized by washing in 70% (v/v) ethanol for 1 min and then in 1% (v/v) sodium hypochlorite for 1 min. The plant tissues were then washed 3–4 times with sterilized distilled water. The leaf tissue pieces were further cut into small pieces (0.2–0.5 cm in length) using a sterile scalpel and placed on PDA medium. The plates were incubated at 25 ± 2°C for 7–10 days, and pure cultures of the endophytic fungi were obtained (Lu et al. 2012).DNA was extracted from the mycelia for molecular identification of endophytic fungi according to the method described by Liu et al. (2000). PCR amplification of the Internal Transcribed Spacer (ITS) regions of the fungal rDNA was performed using the primers ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) as described by Halo et al. (2018). The PCR products of the expected sizes were sequenced at Macrogen, Seoul, Korea. The sequences were subjected to BLAST searches using the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov).A dual culture technique was used to test the in vitro antagonistic effect of the endophytic fungi against M. cannonballus. A mycelial plug (7-mm diameter) was excised from the fungal endophyte colonies and placed on one side of a PDA plate (90-mm diameter) about 1 cm away from the edge. On the same plate, a 7-mm diameter disc of M. cannonballus was placed on the opposite side at 1 cm distance from the edge. The Petri plates inoculated with M. cannonballus alone were used as control. Four Petri plates per treatment were used. The Petri plates were incubated at 25 ± 2°C, and the radial growth of M. cannonballus was measured after 5–7 days of incubation. The mycelial growth inhibition was calculated using the following formula: % inhibition=[1−(T/C)]×100where C – radial growth of M. cannonballus in the control plate and T – radial growth of M. cannonballus in the dual culture plate (Toghueo et al. 2016).To investigate the antagonistic effects of the endophytic fungi on the morphology of M. cannonballus hyphae, the five-mm agar plug samples of M. cannonballus were excised from the colony edges of inhibition zone in the dual culture plate. The samples for scanning electron microscopy were prepared according to the method reported by Goldstein et al. (2003) and observed with a JEOL (Model: JSM-7800F) scanning electron microscope. The culture of M. cannonballus grown in the absence of endophytic fungi served as control.To perform the electrolyte leakage assay, the endophytic fungi were cultured in 200 ml of Czapek Dox broth (static) in 500 ml conical flasks at room temperature (25 ± 2°C) for 14 days, and the culture filtrates were obtained by filtering through Whatman No. 1 filter paper. Five hundred mg of M. cannonballus mycelium were added to 20 ml of culture filtrate in a glass vial. The conductivity of the suspension was measured at 0, 1, and 3 h after incubation by using a conductivity meter (Halo et al. 2018). There were three replicates per treatment and control.Data from the in vitro growth inhibition and the electrolyte leakage assays were statistically analyzed using general linear model ANOVA using Minitab Statistical Software version 17 (Minitab Inc., State College, USA). When ANOVA revealed significant differences between treatments, means were separated using Tukey’s studentized range test at p ≤ 0.05. Arc sine transformation of data on % mycelial growth inhibition was done prior to analysis.A total of five morphologically distinct fungal endophytes were obtained from the leaves of Z. multiflora. Based on the rDNA ITS sequence analysis, these endophytic fungal (Ascomycota, Sordariomycetes) isolates were identified as Nigrospora sphaerica (Amphisphaeriales, Apiosporaceae) (E1 and E6), Subramaniula cristata (Sordariales, Chaetomiaceae) (E7) and Polycephalomyces sinensis (Hypocreales, Ophiocordycipitaceae) (E8 and E10). The sequences were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers {"type":"entrez-nucleotide","attrs":{"text":"MH028052","term_id":"1509265912","term_text":"MH028052"}}MH028052, {"type":"entrez-nucleotide","attrs":{"text":"MH028054","term_id":"1509265914","term_text":"MH028054"}}MH028054, {"type":"entrez-nucleotide","attrs":{"text":"MH028055","term_id":"1509265915","term_text":"MH028055"}}MH028055, {"type":"entrez-nucleotide","attrs":{"text":"MH028056","term_id":"1509265916","term_text":"MH028056"}}MH028056, and {"type":"entrez-nucleotide","attrs":{"text":"MH028058","term_id":"1509265918","term_text":"MH028058"}}MH028058. P. sinensis is an important medicinal fungus. Numerous pharmacological activities of P. sinensis including immunomodulatory, anti-estrogenicity and antitumor activities have been documented (Wang et al. 2012). N. sphaerica has been reported as an endophyte (Wang et al. 2017) as well as a pathogen in a few plant species (Wright et al. 2008; Liu et al. 2016). However, Z. multiflora plants colonized with these endophytic fungi were healthy and did not show any observable disease symptoms.The in vitro dual culture antagonism assay showed that all the five endophytic fungi inhibited the mycelial growth of M. cannonballus. N. sphaerica E1 was the most effective (81.7%), followed by P. sinensis E8 (80.6%), P. sinensis E10 (75.8%) and N. sphaerica E6 (66.1%). S. cristata E7 was the least effective, which recorded 38.7% inhibition (Table ​(TableI,I, Fig. ​Fig.1).1). Further, scanning electron microscopic observations of the hyphae of M. cannonballus from the dual culture assay plates at the edge of the inhibition zone revealed morphological abnormalities such as disintegration, shrinkage, and loss of turgidity. Scanning electron micrograph of M. cannonballus after co-cultivation with the endophytic fungus N. sphaerica E1 is shown in Fig. ​Fig.2.2. These findings corroborate with those of Hajlaoui et al. (1992) who reported plasmolysis of Sphaerotheca pannosa var. rosae mycelium due to the antagonistic effect of Sporothrix flocculosa. Halo et al. (2018) reported shrinkage of Pythium aphanidermatum hyphae due to the antagonistic activity of Aspergillus terreus. The shrinkage of M. cannonballus hyphae in the present study suggests a possible leakage of cytoplasmic contents (Garg et al. 2010). The loss of the turgidity of M. cannonballus hyphae indicates alterations in the permeability of the cell membrane (Halo et al. 2018). Several reports indicate the production of antimicrobial substances by endophytic fungi (Zhao et al. 2012; Homthong et al. 2016). Kim et al. (2001) demonstrated that phomalactone, a compound produced by N. sphaerica restricted the mycelial growth and germination of sporangium and zoospore of Phytophthora infestans and decreased the incidence of late blight in tomato. Zhao et al. (2012) characterized four secondary antifungal metabolites viz., dechlorogriseofulvin, griseofulvin, mullein, and 8-dihydroramulosin from the liquid cultures of the endophytic fungus Nigrospora sp. isolated from roots of the medicinal plant, Moringa oleifera. Homthong et al. (2016) reported the production of chitinase by Paecilomyces (Polycephalomyces) sp. The inhibitory effect of endophytic fungi on the hyphae of M. cannonballus in this study might be due to the production of antifungal metabolites.Table IPercentage inhibition of mycelial growth of M. cannonballus by endophytic fungi isolated from Zataria multiflora in dual cultures on PDA.

Selection of candidate coding DNA barcoding regions for use on land plants

An in silico screen of 41 of the 81 coding regions of the Nicotiana plastid genome generated a shortlist of 12 candidates as DNA barcoding loci for land plants. These loci were evaluated for amplification and sequence variation against a reference set of 98 land plant taxa. The deployment of multiple primers and a modified multiplexed tandem polymerase chain reaction yielded 85–94% amplification across taxa, and mean sequence differences between sister taxa of 6.1 from 156 bases of accD to 22 from 493 bases of matK. We conclude that loci should be combined for effective diagnosis, and recommend further investigation of the following six loci: matK, rpoB, rpoC1, ndhJ, ycf5 and accD. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 1–11.     

3,5-Dichlorophenoxyacetic acid as a growth retardant

Tomato seedlings, grown in the glasshouse, were sprayed with solutions of 3,5-dichlorophenoxyacetic acid as sodium salt at 2 times 10^-5, 4 times 10^-5 and 10^-4 M. The treated plants became dark-green, dwarfed, and compact. After 6–7 weeks normal growth was resumed. Measurements and analytical data on treated and control plants are presented.     

Pennsylvanian phylloid algae interpreted as shallow-water xenophyophores

Xenophyophores, a group of agglutinated rhizopod protists, occur in nearly all the world's oceans at depths generally in excess of 1OOO m. In certain areas they dominate deep-sea, megafaunal communities. Their widespread distribution indicates a well-established adaptive radiation. Only one fossil analogue, however, has been proposed. This is Paleodictyon , a trace fossil that superficially resembles the Recent infaunal xenophyophore Occultammina profunda in morphology. The platy xenophyophore genus Psammina closely resembles certain late Palaeozoic fossils from northeastern Kansas that have been referred to as 'phylloid algae'. Here we compare modern xenophyophore structure with that of the fossil phylloids. By reconstructing the fossils, analysing their thin sections with polarized, blue, and ultraviolet light and subjecting them to Q-switched laser ablation, we conclude that certain phylloids could represent shallow-water xenophyophores. *** Phylloid algae, xenophyophores, motphology, reconstruction, laser ablation, Pennsylvanian.