Studies on sodium bypass flow in lateral rootless mutants lrt1 and lrt2, and crown rootless mutant crl1 of rice (Oryza sativa L.)

An apoplastic pathway, the so‐called bypass flow, is important for Na⁺ uptake in rice (Oryza sativa L.) under saline conditions; however, the precise site of entry is not yet known. We report the results of our test of the hypothesis that bypass flow of Na⁺ in rice occurs at the site where lateral roots emerge from the main roots. We investigated Na⁺ uptake and bypass flow in lateral rootless mutants (lrt1, lrt2), a crown rootless mutant (crl1), their wild types (Oochikara, Nipponbare and Taichung 65, respectively) and in seedlings of rice cv. IR36. The results showed that shoot Na⁺ concentration in lrt1, lrt2 and crl1 was lower (by 20–23%) than that of their wild types. In contrast, the bypass flow quantified using trisodium‐8‐hydroxy‐1,3,6‐pyrenetrisulphonic acid (PTS) was significantly increased in the mutants, from an average of 1.1% in the wild types to 3.2% in the mutants. Similarly, bypass flow in shoots of IR36 where the number of lateral and crown roots had been reduced through physical and hormonal manipulations was dramatically increased (from 5.6 to 12.5%) as compared to the controls. The results suggest that the path of bypass flow in rice is not at the sites of lateral root emergence.     

Root Formation in Pea Cuttings

Cuttings were either decapitated or both decapitated and disbudded at different time intervals. Auxin, at different concentrations, was applied to the cuttings in lanoline. Auxin applied to decapitated and disbudded cuttings promoted root formation in the early stage of the initiation phase. Auxin treated cuttings, which were only decapitated, did not show an increase in number of roots per cutting. However, an increase in the root mass was found in the early stage of the initiation phase. The results seem to indicate that auxin is active only in the first part of the initiation phase. It is acting alone, not together with other substances synthesized in the shoot meristem.     

Erratum to: Associations of POU1F1 gene polymorphisms and protein structure changes with growth traits and blood metabolites in two Iranian sheep breeds

Journal of Genetics -     

Biofloc Technology: Emerging Microbial Biotechnology for the Improvement of Aquaculture Productivity

With the significant increases in the human population, global aquaculture has undergone a great increase during the last decade. The management of optimum conditions for fish production, which are entirely based on the physicochemical and biological qualities of water, plays a vital role in the prompt aquaculture growth. Therefore, focusing on research that highlights the understanding of water quality and breeding systems’ stability is very important. The biofloc technology (BFT) is a system that maximizes aquaculture productivity by using microbial biotechnology to increase the efficacy and utilization of fish feeds, where toxic materials such as nitrogen components are treated and converted to a useful product, like a protein for using as supplementary feeds to the fish and crustaceans. Thus, biofloc is an excellent technology used to develop the aquaculture system under limited or zero water exchange with high fish stocking density, strong aeration, and biota. This review is highlighted on biofloc composition and mechanism of system work, especially the optimization of water quality and treatment of ammonium wastes. In addition, the advantages and disadvantages of the BFT system have been explained. Finally, the importance of contemporary research on biofloc systems as a figure of microbial biotechnology has been emphasized with arguments for developing this system for better production of aquaculture with limited natural resources of water.Key words: biofloc, BFT, aquaculture, microbes, water quality, wastes     

Identification of Surface Antigens of Sarcocystis muris (Coccidia)

Zoites of Sarcocystis muris were recovered from the skeletal muscles of infected mice by trypsin digestion. Extracts of zoites prepared by freeze-thaw, Triton X-100 (0.1%), or a combination of the two treatments contained antigenic components. Testing of these antigens by agar gel diffusion and immunoelectrophoresis against sera from infected mice showed one major precipitin band. SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) of the extracts revealed at least eight detectable polypeptides ranging in molecular weight from 10,000 to 220,000. The antigenic components of the extract were identified by labeling the parasite surface with [¹²⁵I] and precipitation of the [¹²⁵I]-labeled antigens with immune sera. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed three antigens with molecular weights of 27,500, 43,000 and 90,000. The smallest of these was the predominant antigen as suggested by labeling intensity.     

Expression and regulation of Sclerotinia sclerotiorum necrosis and ethylene-inducing peptides (NEPs)

Successful host colonization by necrotrophic plant pathogens requires the induction of plant cell death to provide the nutrients needed for infection establishment and progression. We have cloned two genes encoding necrosis and ethylene-inducing peptides from Sclerotinia sclerotiorum , which we named SsNep1 and SsNep2 . The peptides encoded by these genes induce necrosis when expressed transiently in tobacco leaves. SsNep1 is expressed at a very low level relative to SsNep2 during infection. The expression of SsNep2 was induced by contact with solid surfaces and occurred in both the necrotic zone and at the leading margin of the infection. SsNep2 expression was dependent on calcium and cyclic adenosine monophosphate signalling, as compounds affecting these pathways reduced or abolished SsNep2 expression coincident with a partial or total loss of virulence.     

Effects and modes of action of citrus limonoids against Leptinotarsa decemlineata

ABSTRACT Limonin and its five derivatives were evaluated in feeding bioassays for depression effects on feeding, growth and development of the fourth stadium Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Epilimonol and limonin diosphenol were most effective in depression of feeding and growth, with ED₅₀s 10 and 8 μg/cm². At a dosage of 31.6 μg/cm², epilimonol, limonin diosphenol and limonin caused both significant feeding reduction and developmental delay, while deoxylimonol, tetrahydrolimonin and limonol were not effective. Mouth-part coating and antifeedant bioassays show that feeding inhibition at host acceptance level seems to be the primary mode of action of epilimonol and limonin diosphenol.     

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     

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.