Indirect Inoculation Research Articles

Enhancing Bio-Fertilization Potential of Introduced Hedysarum Species Through Indirect Root Inoculation: Implications for Agro-Ecological Sustainability in Arid Lands

Saharan soils are devoid of Rhizobium bacteria specific to the Sulla plants which is indispensable for nitrogen fixation and soil fertility. The present study aims to test the possibility of formation nodules on the roots of some species of Hedysarum (Sulla) genus introduced in Ghardaïa region with indirect inoculation in the reason to optimize the use of nitrogen fertilizers. Three soil samples were chosen from three different districts of Algeria (Tizi-Ouzou, Sétif and El-Tarf) to be used as substrate and source of bacterial strains for three different species of Sulla (Hedysarum coronarium, Hedysarum carnosum, Hedysarum flexuosum). The results showed the formation of a considerable number of nodules in all treatments. The effect of species and samples of soils on the studied parameter was highly significant (p<0.000) at the 5% significance level. All cultivated species in the soil of Sétif could form significant more nodules compared to other soils. The disparity in the average number of nodules formed in the soil of Sétif represents a rate of 78.75% compared to the soil of Tizi-Ouzou. This study could contribute to improve fodder production of Sulla species cultivated in Ghardaïa region and also provides a forage rich in protein for local livestock.

Corneal Culture in Infectious Keratitis: Effect of the Inoculation Method and Media on the Corneal Culture Outcome.

Background: To compare two different methods of corneal culture in infectious keratitis: multiple sampling for direct inoculation and enrichment (standard method) and a single sample via transport medium for indirect inoculation (indirect inoculation method). Methods: Prospective inclusion of patients fulfilling predefined criteria of infectious keratitis undergoing corneal culture according to both studied methods in a randomized order. Results: The standard method resulted in a significantly higher proportion of positive culture outcomes among the 94 included episodes of infectious keratitis (61%; 57/94) than the indirect inoculation method (44%; 41/94) (p = 0.002) and a significantly higher proportion of microorganisms than the indirect inoculation method, with a Cohen’s kappa of 0.38 (95% CI: 0.28–0.49) for agreement between the methods. Subanalysis of culture results showed that direct inoculation on gonococcal agar only combined with the indirect inoculation method resulted in a similar rate of culture positive patients and proportion of detected microorganisms to the standard method. Conclusion: Indirect inoculation of one corneal sample cannot replace direct inoculation of multiple corneal samples without loss of information. A combination of directly and indirectly inoculated samples can reduce the number of corneal samples by four without statistically significant differences in culture outcome or in the proportion of detected microorganisms.

Pathogenicity of commercial entomopathogenic fungal strains on the avocado seed borer (ASB), Heilipus lauri (Coleoptera: Curculionidae) under laboratory conditions

The avocado seed borer, (ASB) Heilipus lauri Boheman 1845 (Coleoptera: Curculionidae) is an official quarantine pest limiting avocado production and fruit exports from Colombia to pest-free countries. To provide alternatives to manage ASB, the objective of this study was to determine the potential use of commercial strains of Beauveria bassiana and Metarhizium anisopliae as biological control agents of this pest. Pathogenicity of the strains was evaluated by complete immersion of the insect in a spore solution (direct inoculation) or by insect contact with inoculated fruit (indirect inoculation). The B. bassiana strain caused higher mortality (DF = 3, F = 25.7, P = 0.0002), showing an almost eight-fold increase, 55% and 79% mortality, for both concentrations used, 2.5 × 108 spores/mL or 2.5 × 109 spores/mL, respectively. Additionally, the B. bassiana strain showed shorter median survivorship using direct inoculation (15.8 to 8.7 days), a longer delay in the feeding initiation time (50 h), and a decrease in the fruit intake (Anti-feeding index above 50) after indirect inoculation. To our knowledge, this is the first report about the pathogenicity of a commercial B. bassiana strain against ASB in Colombia.

The Potential Risk of Plant-Virus Disease Initiation by Infected Tomatoes.

During 2019, tomato fruits showing viral-like symptoms of marbled yellow spots were abundant in Israel. The new symptoms were distinctive from those typical of tomato brown rugose fruit virus (ToBRFV) infection but resembled symptoms of pepino mosaic virus (PepMV) infection. RT-PCR analysis and the serological tests (enzyme linked immunosorbent assay, western blot and in situ immunofluorescence) revealed and confirmed the presence of both the tobamovirus ToBRFV and the potexvirus PepMV in the symptomatic fruits. A mixture of rod-like and filamentous particles, characteristic of viruses belonging to tobamovirus and potexvirus genera, was visualized by transmission electron microscopy of the tomato fruit viral extract. Sanger sequencing of amplified PepMV-coat protein gene segments showed ~98% sequence identity to the Chilean (CH2)-strain. In a biological assay testing the contribution of traded infected tomatoes to the establishment of tomato plant disease, we applied direct and indirect inoculation modes using Tm-22-resistant tomato plants. The results, assessed by disease symptom development along with serological and molecular analyses, showed that the ToBRFV and PepMV co-infected fruits were an effective inoculum source for disease spread only when fruits were damaged. Importantly, intact fruits did not spread the viral disease. These results added a new factor to disease epidemiology of these viruses.

Differential gene expression in Acromyrmex leaf‐cutting ants after challenges with two fungal pathogens

Social insects in general and leaf-cutting ants in particular have increased selection pressures on their innate immune system due to their social lifestyle and monoclonality of the symbiotic fungal cultivar. As this symbiosis is obligate for both parties, prophylactic behavioural defences against infections are expected to increase either ant survival or fungus-garden survival, but also to possibly trade off when specific infections differ in potential danger. We examined the effectiveness of prophylactic behaviours and modulations of innate immune defences by a combination of inoculation bioassays and genome-wide transcriptomic studies (RNA-Seq), using an ant pathogen (Metarhizium brunneum) and a fungus-garden pathogen (Escovopsis weberi) and administering inoculations both directly and indirectly (via the symbiotic partner). Upon detection of pathogen conidia, ant workers responded by increasing both general activity and the frequency of specific defence behaviours (self-grooming, allo-grooming, garden-grooming) independent of the pathogen encountered. This trend was also evident in the patterns of gene expression change. Both direct and indirect (via fungus garden) inoculations with Metarhizium induced a general up-regulation of gene expression, including a number of well-known immune-related genes. In contrast, direct inoculation of the fungus garden by Escovopsis induced an overall down-regulation of ant gene expression, whereas indirect inoculation (via the ants) did not, suggesting that increased activity of ants to remove this fungus-garden pathogen is costly and involves trade-offs with the activation of other physiological pathways.

Application Anti Microbial Activity Test and Direct Inoculation of Urinary Specimen Test to Increase the Quality of Results and Decrease the Production Cost in Clinical Microbiology Laboratory, Sanglah General Hospital Hospital, Bali-Indonesia

Objective: Urinary tract infection (UTI) is the most common bacterial infection in general practice and in hospitals. Fast and accurate urine culture and sensitivity test are needed for adequate therapy. Anti Microbial Activity test (AMA test) that is used to detect the presence of antibiotics in urine specimens is not commonly used in clinical microbiology laboratories. Some laboratories are still using indirect inoculation technique using enriched media before inoculating onto the agar media. The aim of this research is to compare results of urinary examination of direct inoculation technique with AMA test with indirect inoculation technique without AMA test. Methods : A number of 210 urine specimens were collected in Clinical Microbiology Laboratory at Sanglah General Hospital within a time period between 16 June until 16 July 2009. Results : Antibiotics were detected in 40% of the urinary specimens; whereas 48.1% showed no evidence of UTI, that is negative AMA test and sterile urinary culture or colony growth < 10 5 CFU/ml. Only 11.9% of the specimens indicates urinary tract infections. The examination can be completed within 2-3 days which is shorter than indirect inoculation test which require 5-7 days. Direct inoculation technique can reduce the cost of production three-fold the costs require for an indirect inoculation test. Conclusions: Application of AMA test and direct inoculation technique can give results more rapidly, reliable and useful for clinicians. This also decrease the laboratory’s cost of production.

Optimizing modes of inoculation of Rhipicephalus ticks (Acari: Ixodidae) with a mitosporic entomopathogenic fungus in the laboratory

The process of strain selection is an important step in the development of insect pathogens for biological control. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using Rhipicephalus pulchellus Gerstäcker (Acari: Ixodidae) as a model. Initially, an oil-based formulation of Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales) titred at 10(9) conidia ml(-1) was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon's spray tower (52.3% tick mortality), although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 x 10(4) +/- 1.1 x 10(3) conidia ml(-1)) after spraying by Burgerjon's spray tower compared to 1 x 10(6) conidia ml(-1) obtained with the microapplicator. Thus, inoculation by Burgerjon's spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC(50) values following contamination of nymphs (LC(50) = 1.4 x 10(7) conidia ml(-1)) and adults (LC(50) = 6.7 x 10(7) conidia ml(-1)) in SS were significantly lower compared to SP; nymphs (LC(50) = 5.7 x 10(8) conidia ml(-1)) and adults (LC(50) = 5.3 x 10(9) conidia ml(-1)) and SW; nymphs (LC(50) = 5 x 10(8) conidia ml(-1)). Although the LC(50) value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at 2 weeks post-treatment) and (23.3% at 3 weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8%, respectively) and SW (0.0 and 0.0). Results show that among the modes of inoculation tested, SP was the most appropriate for inoculating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil.

Mortality of the Nut-Leaf Weevil Strophosoma Melanogrammum (Forster) and Damage Rate of Needles After Treatment with Entomopathogenic Fungi

The susceptibility of the nut-leaf weevil, Strophosoma melanogrammum (Forster) (Coleoptera; Curculionidae) to the select- ed strains of Beauveria bassiana (Bals.-Criv.) Vuillemin, Isaria farinosa (Holm) Brown & Smith, I. fumosorosea (Wize) Brown & Smith, Metarhizium anisopliae (Metschnikoff) Sorokin and M. flavoviride Gams & Rozsypal, was tested under laboratory conditions. Two ways of insect exposure to fungus were studied; direct inoculation of weevils and indirect by contact with inoculated A. procera twigs that served as food for S. melanogrammum. All of the tested isolates were pathogenic to S. melanogrammum. Direct inoculation of adult weevils in fungal spore suspensions resulted in the greatest mortality compared to indirect inoculation where A. procera twigs were immersed in spore suspensions. Direct inoculation with B. bassiana and Metarhizium spp. isolates caused over 80% mortality after 28 days at 20°C, while LT 50 values ranged from 15.0 to 21.6 days. Indirect inoculation with the most effective isolate M. anisopliae (275) resulted in 72% mortality after 28 days with a LT 50 value of 22.1 days. Both direct and indirect spore application failed to reduce needle damage compared with the control. However, significant differences between particular isolates have appeared.

Evaluation of capsular and acapsular strains of S. aureus in an experimental brain abscess model

Brain abscesses are mainly caused by either direct or indirect inoculation of gram positive bacteria including Stapylococcus aureus ( S. aureus) or Streptococcus species into the central nervous system. In the present study, we aimed to compare potential changes in brain abscess pathogenesis induced by two different strains of S. aureus, namely the laboratory strain RN6390 and the clinical isolate Reynolds. Although the Reynolds strain was expected to be more resistant to eradication by the host, due to the existence of a polysaccharide capsule, and subsequently to be more virulent, instead we found parenchymal damage and mortality rates to be more prominent following RN6390 infection. In contrast, the Reynolds strain proliferated faster and induced early expression of the chemokine CXCL2, matrix metalloproteinase-9 (MMP-9), and complement 3a and C5. Furthermore, there were early and more abundant infiltration of PMNs, T cells and erythrocyte extravasation in brain abscesses induced by the Reynolds strain. However, several immune parameters were not different between the two strains during the later stages of the disease. These results suggest that capsular S. aureus can modulate innate immunity and complement system activation differently than the acapsular strain RN6390, and the early changes induced by Reynolds strain may have an important impact on survival.

Reliability of Transport Medium in the Laboratory Evaluation of Corneal Ulcers

To compare the microbiological yield of corneal ulcer cultures established by direct inoculation of culture media vs indirect inoculation by means of transport medium (Amies without charcoal). Single masked, prospective clinical trial. Scrapings were obtained for Gram and potassium hydroxide (KOH) stains from eyes with presumed infectious keratitis and cultured by direct plating onto standard media. Samples were also held in transport media (Amies without charcoal) at room temperature and then plated after 4 and 24 hours. Yields from direct plating vs cultures by means of transport media were compared. Of 100 consecutive eyes examined with presumed infectious keratitis, Gram or KOH stain revealed a bacterial or fungal agent in 69 cases (69%). Of these, 26 were bacterial and 43 fungal. Twenty-two bacterial infections produced positive cultures by direct plating, and all produced the same organism with Amies medium after 4 and 24 hours, respectively. For 43 fungal infections identified by KOH stain, 29 (67%) yielded a positive result after 4 hours in Amies transport medium and 27 (63%) after 24 hours in Amies medium. A total of three cases (7%) that showed fungal infection on KOH stain but did not yield organisms by direct plating did so after inoculation with Amies transport medium. For all comparisons, there was no difference in recovery rates by means of transport medium compared with direct plating (McNemar exact P > .05). In the clinical setting, Amies transport medium may be a useful alternative to direct inoculation onto blood agar for the laboratory evaluation of infectious keratitis.

Safety evaluation of single-use medical devices after submission to simulated reutilization cycles.

Pyrogenic and toxic reactions, especially in immunologically compromised patients, are among the risks associated with reuse of single-use medical devices (SUDs) with recurrent damaged surfaces. These drawbacks have raised serious doubts about the true benefits of the reprocessing practice. Taking into consideration prolonged patients' stay in hospitals due to adverse reactions provoked by recycled SUDs, the safety of these reprocessed materials was evaluated. The reprocessing cycles were simulated after intentional contamination of selected test material such as intravenous catheters, 3-way stopcocks, and tracheostomy tubes with Bacillus subtilis var. niger ATCC 9372 spores (10(7) CFU/unit). The repeated reprocessing cycles consisted of subsequent wash with enzymatic detergent followed by drying and sterilization with ethylene oxide-CFC (12 + 88), 600 mg/L at 55 degrees C, relative humidity 60%, for 3 h. After each reprocessing cycle, specimen samples were evaluated by pour plate microbial counts, direct and indirect inoculation sterility tests, cytotoxicity evaluation, and scanning electron microscopy (SEM). Microbial counts as high as 10(3) CFU were evident even after the 10th reprocessing cycle, besides scratched and damaged surfaces observed by SEM. Risk-benefit viewpoints are discussed.

BIOLOGICAL VALIDATION OF TOMATO PULP CONTINUOUS HEAT PROCESS

ABSTRACT This research validated the commercial process applied to tomato pulp (pH 4.3 and 8 Brix) packed in Tetra Brik packages. Spores of Bacillus coagulans and Neosartorya fischeri were selected as targets. The heat resistance of both microorganisms, tested independently, was compared. The redesigned thermal processes were carried out in a aseptic processing and tested by indirect inoculation and retrieval with spores immobilized in alginate/tomato balls. The results showed that processes for 30 s at 115C or greater did not allow the survival of heat‐resistant molds. For bacterial spores, processes for 30 s at 109C or greater showed no survivors. Although, 30 s at 115C will control both types of spoilage spores, concern for possible C. botulinum growth attributed to metabiosis in product with varying initial populations of molds and residual oxygen content dictated, a process recommendation of 60 s at 126C for safety reasons.

Epidemiology of hepatitis B in Europe and worldwide.

Hepatitis B virus (HBV) infection is a major public health problem and cause of infectious disease mortality worldwide. Approximately 2 billion people one third of the world’s population have serologic evidence of past or present HBV infection, and 350 million people are chronically infected. Each year over 1 million people die from HBV-related chronic liver disease, including cirrhosis and hepatocellular carcinoma (HCC) (1). HCC is one of the most common cancers worldwide, and HBV is responsible for at least 75% of these cancers (2).

Effect of aromatic plant species on vesicular-arbuscular mycorrhizal establishment in Pistacia terebinthus

The inoculation of Pistacia terebinthus with vesicular-arbuscular mycorrhizal (VAM) fungi and the spread of the infection were studied using a mixed cropping system, under glasshouse conditions, with Salvia officinalis, Lavandula officinalis and Thymus vulgaris colonized by Glomus mosseae as an inoculation method. This method was compared with soil inoculum placed under the seed or distributed evenly in the soil. Indirect inoculation with all the aromatic plants tested significantly increased VAM root colonization of P. terebinthus compared with the use of soil inoculum, although the effect on plant growth was different for each one of the aromatic species used as inoculum source. Inoculation with L. officinalis and T. vulgaris were the best treatments resulting in high VAM colonization and growth enhancement of P. terebinthus.

Redistribution of foliar surface deposits of prochloraz by simulated rainfall and the control of eyespot disease of winter wheat

Outdoor-grown wheat plants sown in troughs and infected through indirect inoculation with the eyespot pathogen ( Pseudocercosporella herpotrichoides) were sprayed with prochloraz in a spray chamber at Zadok GS 37. Prochloraz was applied in either low (151/ha) or normal (2001/ha) spray volumes to plants standing vertically or laid on their sides and masked to preclude spray deposition on the stem bases. After spraying, plants were kept in a glasshouse and a proportion were exposed to one or more light (0·4 mm total) or heavy (4·0 mm total) artificial rainfall regimens. Immediately after spraying with recommended amounts of fungicide, only traces were found on stem bases of plants sprayed vertically and, in the absence of rain, eyespot incidence was unaffected. Heavy rain applied one day after fungicide application redistributed from the foliage to the plant base c. 5·5 μg prochloraz/g stem tissue and decreased eyespot severity. Light rain redistributed less fungicide and had a smaller effect on eyespot. Spray volume had no effect on redistribution or disease control. The half-life of prochloraz on unweathered foliage was c. 6 days, but at the stem base, deposits changed little over two weeks.

Vertical Transmission Status of Hepatitis B Virus in Asymptomatic Carriers in Bangladesh – Experience from a Tertiary Centre

doi: 10.3329/taj.v21i1.3209 TAJ 2008; 21(1)