Mechanism and synchronicity of wheat (Triticum aestivum) resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1

Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40 percent by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9 percent confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P

Antimicrobial Potential and Phytochemical Screening of Clathria sp. 1 and Tedania (Tedania) stylonychaeta Sponge Crude Extracts Obtained from the South East Coast of South Africa

Scientists have continuously searched for novel bioactive compounds to overcome the inherent problems associated with drug resistance, the evolution of unknown diseases, and the toxicity of currently used compounds. The ocean has been considered a rich source of compounds that possess unique chemical structures and novel biological capabilities. Biologically active molecules isolated from marine flora and fauna have shown significant advancement over the past century in the pharmaceutical industry. Marine natural products (MNPs) have been used as nanomedicine, cosmetics, wound healing, antimicrobial agents, anticancer agents, and anti-inflammatory agents. The physicochemical parameters of the collection site were also recorded. This study’s marine sponge species were collected from Phillip’s Reef, South Africa, at 12 m during the spring season. Ethyl acetate (EA) and dichloromethane : methanol (DCM : ME, 1 : 1) were used as extraction solvents. Crude extracts of the marine sponges were tested against MRSA, P. aeruginosa, C. difficile, A. fumigatus, and C. albicans. Phytochemical screening was conducted to identify seven critical phytochemical groups. A pH reading of 8.01 and a temperature of 15.45°C were recorded at the sampling site. Clathria sp. 1 and Tedania (Tedania) stylonychaeta EA crude extracts showed bioactivity against all five test pathogens. The DCM : ME crude extract of Clathria sp. 1 was the only bioactive crude extract from DCM : ME extracts. This crude extract was only bioactive against C. albicans as no activity was observed against the other four pathogens. EA crude extracts of Clathria sp. 1 yielded more significant inhibition zones against both fungal pathogens. These EA crude extracts performed better than fluconazole as inhibition zones of 35±0 mm at 24 mg/ml, 31±0 mm at 19 mg/ml, 31±0 mm at 14.4 mg/ml, 30±0 mm at 9.6 mg/ml, and 25±0 mm at 7.2 mg/ml were recorded. Clathria sp. 1 crude extracts exhibited higher inhibition zones compared to Tedania (Tedania) stylonychaeta. The antibiotic imipenem (26±0.7 mm at 10 μg) and ciprofloxacin (30±0.3 mm at 5 μg) exhibited higher zones of inhibition than EA crude extracts of Tedania (Tedania) stylonychaeta at all test concentrations. In this study, Clathria sp. 1 was observed to have broad-spectrum bioactivity as EA crude extracts were bioactive against MRSA, P. aeruginosa, C. difficile, A. fumigatus, and C. albicans. In addition to this, the EA crude extract of Clathria sp. 1 was bacteriostatic (9.6 mg/ml). Clathria sp. 1 DCM : ME crude extract only tested positive for the presence of terpenoids. In contrast, EA crude extracts did not test positive for the existence of any of the seven phytochemicals. Our study has revealed that Tedania (Tedania) stylonychaeta and Clathria sp. 1 sponge species collected from Phillip’s Reef in South Africa can produce bioactive compounds useful against bacterial and fungal species

Occurrence of Virulence Genes Associated with Human Pathogenic Vibrios Isolated from Two Commercial Dusky Kob (Argyrosmus japonicus) Farms and Kareiga Estuary in the Eastern Cape Province, South Africa

Background: Seafood-borne Vibrio infections, often linked to contaminated seafood and water, are of increasing global public health concern. The aim of this study was to evaluate the prevalence of human pathogenic vibrios and their associated virulence genes isolated from fish and water samples from 2 commercial dusky kob farms and Kareiga estuary, South Africa. Methods: A total of 200 samples including dusky kob fish (n = 120) and seawater (n = 80) were subjected to Vibrio screening on thiosulfate-citrate-bile salts-sucrose agar (TCBS). Presumptive isolates were confirmed and delineated to V. cholerae, V. parahaemolyticus, V. vulnificus, and V. fluvialis by PCR. Various pathogenic gene markers were screened: V. parahaemolyticus (trh and tdh), V. vulnificus (vcgE and vcgC) and V. fluvialis (stn, vfh, hupO, vfpA). Restriction Fragment Length Polymorphism (RFLP) of the vvhA gene of V. vulnificus strains was performed to determine the associated biotypes. Results: Total Vibrio prevalence was 59.4% (606/1020) of which V. fluvialis was the most predominant 193 (31.85%), followed by Vibrio vulnificus 74 (12.21%) and V. parahaemolyticus 33 (5.45%). No V. cholerae strain was detected. One of the V. parahaemolyticus strains possessed the trh gene 7 (9.46%) while most (91.9%; 68/74) V. vulnificus isolates were of the E-type genotype. V. fluvialis virulence genes detected were stn (13.5%), hupO (10.4%) and vfpA (1.0%). 12.16% (9/74) of V. vulnificus strains exhibited a biotype 3 RFLP pattern. Conclusions: This is the first report of potentially pathogenic vibrios from healthy marine fish in the study area, and therefore a public health concern

Seasonal Variation in Antimicrobial Activity of Crude Extracts of Psammaplysilla sp. 1 from Phillips Reef, South Africa

Marine invertebrates constitute a diverse group of marine organisms beneficial to humanity due to their therapeutic significance. The marine sponge species Psammaplysilla sp. 1 was collected from Philips Reef, South Africa, over a four-season period and assayed for antimicrobial potential. The physicochemical parameters of the collection site were also recorded. The sponge crude extracts’ antimicrobial activity was evaluated using an agar well diffusion assay against 5 pathogens. Phytochemical screening was conducted to identify the presence of 7 critical phytochemical groups. During the four seasons, the mean water temperature was 17.35°C ± 2.06, with autumn recording the highest (20°C) temperature. Antifungal activity was observed by Psammaplysilla sp. 1 (30 mm) against C. albicans, and this was higher than that showed by standard drugs ICZ-10 µg (15 ± 0.1 mm), FLU-15 µg (21 ± 0.2 mm), and VCZ-5 µg (17 ± 0.1 mm), respectively. Similar bioactivities were observed seasonally with Psammaplysilla sp. 1 (22 mm and 24 mm) during autumn and spring, respectively, against C. difficile while only crude extracts collected in spring showed bioactivity against C. albicans. Psammaplysilla sp. crude extracts showed broad-spectrum bioactivity against all test pathogens. DCM : ME crude extracts tested positive for the presence of 2/7 of the phytochemicals (terpenoids and flavonoids). GC-MS revealed several previously reported biologically active compounds such bicyclo[4.2.0]octa-1,3,5-trien-7-ol and phenol, 2,6-dibromo, some of which have been found in plants. This study revealed that sponge bioactivity is dependent on the season and further validated the antimicrobial potential of South African marine sponges

Antimicrobial Susceptibility of Staphylococcus aureus Isolated from Recreational Waters and Beach Sand in Eastern Cape Province of South Africa

Background: Resistance of Staphylococcus aureus to commonly used antibiotics is linked to their ability to acquire and disseminate antimicrobial-resistant determinants in nature, and the marine environment may serve as a reservoir for antibiotic-resistant bacteria. This study determined the antibiotic sensitivity profile of S. aureus isolated from selected beach water and intertidal beach sand in the Eastern Cape Province of South Africa. Methods: Two hundred and forty-nine beach sand and water samples were obtained from 10 beaches from April 2015 to April 2016. Staphylococcus aureus was isolated from the samples using standard microbiological methods and subjected to susceptibility testing to 15 antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) was detected by susceptibility to oxacillin and growth on Brilliance MRSA II agar. Antibiotic resistance genes including mecA, femA rpoB, blaZ, ermB, ermA, ermC, vanA, vanB, tetK and tetM were screened. Results: Thirty isolates (12.3%) were positive for S. aureus by PCR with over 50% showing phenotypic resistance to methicillin. Resistance of S. aureus to antibiotics varied considerably with the highest resistance recorded to ampicillin and penicillin (96.7%), rifampicin and clindamycin (80%), oxacillin (73.3%) and erythromycin (70%). S. aureus revealed varying susceptibility to imipenem (96.7%), levofloxacin (86.7%), chloramphenicol (83.3%), cefoxitin (76.7%), ciprofloxacin (66.7%), gentamycin (63.3%), tetracycline and sulfamethoxazole-trimethoprim (56.7%), and vancomycin and doxycycline (50%). All 30 (100%) S. aureus isolates showed multiple antibiotic-resistant patterns (resistant to three or more antibiotics). The mecA, femA, rpoB, blaZ, ermB and tetM genes were detected in 5 (22.7%), 16 (53.3%), 11 (45.8%), 16 (55.2%), 15 (71.4%), and 8 (72.7%) isolates respectively; Conclusions: Results from this study indicate that beach water and sand from the Eastern Cape Province of South Africa may be potential reservoirs of antibiotic-resistant S. aureus which could be transmitted to exposed humans and animals