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Listeria monocytogenes causes relatively few outbreaks linked to whole fresh produce but triggers recalls each year in the United States. There are limited data on the influence of wet versus dry inoculation methods on pathogen growth on whole produce. A cocktail of five L. monocytogenes strains that included clinical, food, and environmental isolates associated with foodborne outbreaks and recalls was used. Cultures were combined to target a final wet inoculum concentration of 4 to 5 log CFU/mL. The dry inoculum was prepared by mixing wet inoculum with 100 g of sterile sand and drying for 24 h. Produce investigated belonged to major commodity families: Ericaceae (blackberry, raspberry, and blueberry), Rutaceae (lemon and mandarin orange), Rosaceae (sweet cherry), Solanaceae (tomato), Brassaceae (cauliflower and broccoli), and Apiaceae (carrot). Whole intact, inoculated fruit and vegetable commodities were incubated at 2, 12, 22, and 35 ± 2°C. Commodities were sampled for up to 28 days, and the experiment was replicated six times. The average maximum growth increase was obtained by measuring the maximum absolute increase for each replicate within a specific commodity, temperature, and inoculation method. Data for each commodity, replicate, and temperature were used to create primary growth or survival models describing the lag phase and growth or shoulder and decline as a function of time. Use of a liquid inoculum (versus dry inoculum) resulted in a markedly increased L. monocytogenes growth rate and growth magnitude on whole produce surfaces. Temperature highly influenced this difference: a greater effect seen with more commodities at higher temperatures (22 and 35°C) versus lower temperatures (2 and 12°C). These findings need to be explored for other commodities and pathogens. The degree to which wet or dry inoculation techniques more realistically mimic contamination conditions throughout the supply chain (e.g., production, harvest, postharvest, transportation, or retail) should be investigated.

Beauveria bassiana , an entomopathogenic fungus, can exist asymptomatically as an endophyte in many plants. This study aimed to determine the efficiency of B. bassiana colonization of tomato plants using different inoculation methods, and how colonization of the host plant affects the key pest of tomato, the whitefly Bemisia tabaci . To confirm fungal colonization efficiency, nested PCR technique was used to detect B. bassiana sequences in Solanum lycopersicum . Distribution of the fungus within the plant parts was determined by selective medium. Tomato growth parameters were determined with plants that had been inoculated using direct spraying or irrigation of the rhizosphere. B. tabaci performance assays were carried out in a cage, and preference to treatments was determined using Y-tube olfactometer studies. Results show that B. bassiana can effectively colonize tomato, with colonization rate using leaf spraying reaching 100% within 14 days. Fungal presence was not uniformly distributed among plant parts, but was biased towards the inoculation sites. For inoculation, conidial suspension sprayed at 1 × 10 8 conidia/ml resulted in the highest number of isolated colonies in leaf tissue, 8.5 ± 2.02 colonies per 2 g of fresh tissue ( F 4,19  = 2.779, P  = 0.045), and reached the lowest with root treatment. Although only small differences were observed among the growth indicators, leaf spray inoculation resulted in a significant positive influence on plant growth (PC1 55.7% contributions scores = 2.645) in further Principal component analysis. As for the feeding selectivity, B. tabaci preferred uninoculated plants. In Y-tube olfactometer assay, 80.5% of adults selected uninoculated plants. These findings add to the understanding of the interactions between B. bassiana and plants and indicate the potential of expanding the use of entomopathogenic fungi for crop protection.

The effects of the inoculation method of Saccharomyces bayanus BV818 and non-Saccharomyces yeast Metschnikowia agaves P3-3 and the fermentation temperature on the volatile profiles of red pitaya wine were investigated in the present study. Although the growth of P3-3 was inhibited by BV818 in the mixed inoculations, simultaneous and sequential inoculations promoted the production of seven volatiles, including higher alcohols (propan-1-ol, 3-methyl-1-butanol and phenethyl alcohol), esters (ethyl decanoate and diethyl succinate), acid (2-ethylhexanoic acid), and ketone (acetoin). Sequential inoculation produced the largest total content of volatile compounds and exhibited the best in the global aroma. The red pitaya wine produced in different inoculations can be separated by its main volatile components. Furthermore, the highest total content was yielded at 25 °C for alcohols and at 21 °C for esters and acids. Within an experimental range of 17 °C to 29 °C, the contents of benzaldehyde and acetoin decreased with the increase in temperature, whereas the change in 4-ethyl-2-methoxyphenol content was the opposite. The similarly high total contents of volatiles and global aroma score were yielded via sequential inoculation at 21 °C and 25 °C. Therefore, the desired red pitaya wine can be effectively produced by modulating the inoculation method and fermentation temperature.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease requiring hospitalization in infants. There are no market-approved vaccines or antiviral agents available, but a growing number of vaccines and therapeutics are in (pre)clinical stages of development. Reliable animal models are crucial to evaluate new vaccine concepts, but in vivo RSV research is hampered by the lack of well-characterized animal models that faithfully mimic the pathogenesis of RSV infection in humans. Mice are frequently used in RSV infection and vaccination studies. However, differences in the use of mouse strains, RSV subtypes, and methodology often lead to divergent study outcomes. To our knowledge, a comparison between different RSV inoculation methods in mice has not been described in the literature, even though multiple methods are being used across different studies. In this study, we evaluated various pathological and immunological parameters in BALB/c mice after intratracheal or intranasal inoculation with RSV-A2. Our study reveals that intranasal inoculation induces robust pathology and inflammation, whereas this is not the case for intratracheal inoculation. As immunopathology is an important characteristic of RSV disease in infants, these data suggest that in mice intranasal inoculation is a more appropriate method to study RSV infection than intratracheal inoculation. These findings will contribute to the rational experimental design of future in vivo RSV experiments.

The pathogenicity of grapevine geminivirus A (GGVA), a recently identified DNA virus, to grapevine plants remains largely unclear. Here, we report a new GGVA isolate (named GGVAQN) obtained from grapevine ‘Queen Nina’ plants with severe disease symptoms. The infectious clone of GGVAQN (pXT-GGVAQN) was constructed to investigate its pathogenicity. Nicotiana benthamiana plants inoculated with GGVAQN by agroinfiltration displayed upward leaf curling and chlorotic mottling symptoms. A simple, quick, and efficient method for delivering DNA clones of GGVAQN into grapevine plants was developed, by which Agrobacterium tumefaciens cells carrying pXT-GGVAQN were introduced into the roots of in vitro-grown ‘Red Globe’ grape plantlets with a syringe. By this method, all ‘Red Globe’ grape plants were systemically infected with GGVAQN, and the plants exhibited chlorotic mottling symptoms on their upper leaves and downward curling, interveinal yellowing, and leaf-margin necrosis symptoms on their lower leaves. Our results provide insights into the pathogenicity of GGVA and a simple and efficient inoculation method to deliver infectious viral clones to woody perennial plants.

Complete range of symptoms of bacterial blight caused by Xanthomonas axonopodis pv. punicae on various pomegranate plant parts including fruit are described and illustrated. Some of the symptoms are reported for the first time. The pathogen caused blight symptoms on all plant parts except roots and flower. Among nine different inoculation methods evaluated for X. axonopodis pv. punicae on pomegranate, the spray method convincingly proved that spraying of inoculum yielded highly reproducible symptoms within 21 days under optimal temperature and humidity conditions and up to 39 days under less favourable conditions. Spray method was found to show blight symptoms on leaves with incidence of 71.0% and severity of 55.5%. Besides, the lesions which appeared on plants mimicked the natural symptoms observed in orchards. The incubation period for blight development ranged from 4 to 19 days depending on temperature and humidity conditions. The most favorable condition for onset of blight as well as completion of disease cycle was when temperature ranged between 25-35°C for at least 16h and relative humidity >30% for 24h. The spray method of inoculation is, therefore, recommended as a rapid, reproducible screening technique for pomegranate genotypes in resistance breeding programmes. The study indicated that X. axonopodis pv. punicae infects plant tissues through both natural opening as well as by injuries. The culture and PCR based detection assay further confirmed that the bacterial blight pathogen displayed no translaminar systemic movement and caused only localized lesions.

Blackcurrant reversion virus (BRV) is the most destructive currant-infecting and mite-transmitted pathogen from the genus Nepovirus. In this work, BRV transmission in the system Ribes ex vitro–Ribes in vitro was applied for the first time. Triple infection of BRV identified in blackcurrant cv. Gojai was used for phylogenetic analysis and inoculation assay. Transmission of BRV was successful due to its stability in the inoculum for up to 8 days at 4 °C; all BRV isolates were infectious. Our suggested inoculation method through roots was applied in six Ribes spp. genotypes with 100.0% reliability, and the expression levels of defence-related gene PR1 to biotic stress was observed. The prevalence of the virus in microshoots after 2–14 days post-inoculation (dpi) was established by PCR. In resistant genotypes, the BRV was identified up to 8 dpi; meanwhile, infection remained constant in susceptible genotypes. We established that BRV transmission under controlled conditions depends on the inoculum quality, post-inoculation cultivation temperature, and host-plant susceptibility to pathogen. This in vitro inoculation method opens possibilities to reveal the resistance mechanisms or response pathways to BRV and can be used for the selection of resistant Ribes spp. in breeding programs.

Microdochium majus and M. nivale are the primary agents of Fusarium seedling blight (FSB) of wheat in wet and cool crop growing regions. The differential responses and plant traits (root and stem length) of eleven wheat cultivars were evaluated using three inoculation methods with M. majus or M. nivale in soil, potato dextrose agar plate or detached leaf assay to develop a reliable screening method for the identification of resistance to FSB caused by Microdochium species in wheat. Microdochium nivale was the more aggressive FSB pathogen causing 30 % higher disease severity than M. majus and impacting significantly on root length and stem length. In contrast, M. majus was more pathogenic than M. nivale on wheat leaves reducing significantly the maximum efficiency of photosystem (PS) II (Fv’/Fm’). Fv’/Fm’ provides an estimate of the efficiency of PSII photochemistry (photosynthesis) at a given photosynthetically-active photon flux density defined as PSII operating efficiency if all PSII reaction centres were ‘open’. Regression analysis suggested that reduction in stem and/or root length and Fv’/Fm’ can be used as indicators of disease severity and for the detection of tolerance in wheat cultivars to specific diseases in the Fusarium complex. There were significant interactions between genotypes and species for the assessed disease and plant traits suggesting that resistance/tolerance mechanisms and genes maybe different to disease caused by individual Microdochium species. The most resistant cultivar to FSB identified consistently using all methods was Petrus, also known to be resistant to Fusarium head blight suggesting that this cultivar may possess useful durable resistance to more than one disease in the Fusarium disease complex.

A major current challenge is to increase the food production while preserving natural resources. Agricultural practices that enhance the productivity and progressively improve the soil quality are relevant to face this challenge. Trichoderma species are widely used in agriculture to stimulate the plant growth and to control different pathogens affecting crops, representing useful tools for sustainable food production. This mini-review summarizes applications of Trichoderma strains in agriculture to control fungal pathogens, nematodes and insects, the involved biocontrol mechanisms, efficacy and inoculation forms in greenhouse, field and post-harvest conditions. Aspects of Trichoderma handling that influence on biocontrol efficacy such as preventive treatments, frequency of applications and delivery methods are discussed. Strategies useful to improve the antagonistic performance such as the use of native strains, protoplast fusion, formulation, growth on pathogen cell wall medium and combination with other antagonists in integrated treatments are discussed. This mini-review provides practical knowledge to design safe and optimal biocontrol strategies based on Trichoderma and pose challenges to expand its antagonist performance.

The development and optimization of protocols for the precise and pre-symptomatic detection of diseases, and non-invasive evaluation of genotype-specific pathogen resistance enabling selection of the more promising genotypes in breeding programmes are important and often overlooked topics in precision agriculture. The increasing pressure to minimize both production costs and the environmental impact of pesticides forces the search for rapid and objective methods of screening pathogen resistance. Using the non-destructive pulse amplitude modulated (PAM) chlorophyll fluorescence imaging technique, we hypothesized that not only disease detection but also discrimination between differences in the level of resistance of wheat cultivars to the leaf rust (Puccinia triticina Erics.) pathogen can be achieved. Experiments were conducted using the cultivars Dekan and Retro as representatives of a susceptible and a highly resistant genotype, respectively. Fluorescence measurements were carried out daily on the control and on plants inoculated with P. triticina until the first small red-brown pustules appeared in the centre of chlorotic spots. In response to pathogen inoculation, the fluorescence readings showed an early characteristic increase in Y(NO) in both resistant and susceptible cultivars. The susceptible cultivar, however, showed a more pronounced difference between Y(NO) values measured on the control and inoculated leaves as well as a distinct evolution over time. Accordingly, our results indicate that Y(NO) might be suitable for discriminating between wheat genotypes as early as 2 days after inoculation. Thus, the proposed protocol might be adopted as an additional tool for the early screening of new genotypes, especially in breeding programs that aim for high resistance to disease and low crop variability for precision agriculture. However, its implementation in experimental field plots requires improvement of the measurement system and establishment of appropriate algorithms for disease pattern recognition and data analysis.