DNA-based quantification of Fusarium oxysporum f. sp. vasinfectum in environmental soils to describe spatial variation in inoculum density

Fusarium wilt of cotton, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (FOV), occurs in regions of the United States where cotton (Gossypium spp.) is grown. Race 4 of this pathogen (FOV4) is especially aggressive and does not require the co-occurrence of the root knot nematode (Meloidogyne incognita) to infect cotton. Its sudden appearance in far-west Texas in 2016 after many years of being restricted to California is of great concern, as is the threat of its continued spread through the cotton-producing regions of the United States. The aim of this research was to analyze the spatial variability of FOV4 inoculum density in the location where FOV4 is locally emerging, using quantitative and droplet digital polymerase chain reaction (qPCR and ddPCR) methods. Soil samples collected from a field with known FOV4 incidence in Fabens, Texas were analyzed. Appreciable variation in inoculum density was found to occur at spatial scales smaller than the size of plots involved in cultivar trial research, and was spatially autocorrelated (Moran’s I, Z = 17.73, p < 0.0001). These findings indicate that for cultivar trials, accounting for the spatial distribution of inoculum either by directly quantifying it or through the use of densely-distributed “calibration checks” is important to the interpretation of results.

Factors affecting the fitness of an entomopathogenic nematode (Heterorhabditis bacteriophora SUP strain) for use in biocontrol in a horticultural area of Argentina

Summary The optimum conditions for storage, infectivity and multiplication of the nematode Heterorhabditis bacteriophora SUP strain, isolated from horticultural soils in Argentina, were determined for later use in biocontrol. This strain was able to kill Tenebrio molitor larvae (over 90%) at 16 and 25°C, after 7 and 4 days, respectively, with mortality being significantly higher at 25°C. No infectivity was reported at 4 and 11°C. Higher concentrations (over 150 infective juveniles (IJ) insect−1) produced higher percentages of mortality. However, increasing inoculum density reduced offspring production and increased emergence time. Storage density between 2500 and 7500 IJ ml−1 had no effect on survival of IJ. Survival of IJ maintained at 4 and 16°C was around 80% after 4 weeks storage and 30% after 32 weeks. However, at 25°C, a 60% IJ survival was observed at 4 weeks, decreasing to around 10% after 16 weeks and with no survival at 24 weeks. An overall significant decrease in virulence was observed after 90 and 120 days of storage but this was more pronounced at a storage temperature of 4°C than at 16 and 25°C. IJ stored at 4°C had reduced virulence after 90 and 120 days, but not after 60 days. This study provides useful information about some fundamental environmental conditions influencing important nematode traits of the species H. bacteriophora.

Plasmodiophora brassicae Inoculum Density and Spatial Patterns at the Field Level and Relation to Soil Characteristics

Clubroot, caused by Plasmodiophora brassicae, is an important soilborne disease of the Brassicaceae. Knowledge of the spatial dynamics of P. brassicae at the field level and the influence of soil properties on pathogen spatial patterns can improve understanding of clubroot epidemiology and management. To study the spatial patterns of P. brassicae inoculum density and their relationship to different soil properties, four clubroot-infested fields in central Alberta, Canada, were sampled in 2017 and 2019, and P. brassicae inoculum density, soil pH, and boron, calcium, and magnesium concentrations were quantified. Spatial autocorrelation of the inoculum density was estimated for each of the fields in both years with the Moran’s I and semi-variograms. A Bayesian hierarchical spatial approach was used to model the relationship between P. brassicae inoculum density and the soil parameters. Patchiness of the pathogen was detected, with most patches located at the field edges and adjacent to the entrance. Infested patches grew in size from 2017 to 2019, with an average increase in diameter of 221.3 m and with this growth determined by the maximum inoculum density and active dispersal methods such as movement by machinery and wind. Soil pH, boron, calcium, and magnesium concentrations were not found to have an important effect on the inoculum density of P. brassicae.

Optimization of culture conditions for in vitro adventitious roots and selected flavonoids production in Boesenbergia rotunda liquid suspension culture

Boesenbergia rotunda (L.) Mansf. is one of the unique monocotyledonous perennial plant species belonging to the ginger (Zingiberaceae) family. Locally known as ‘Temu Kunci’ in Malaysia and Indonesia, this medicinal plant has been widely used in Asian dishes, particularly as a condiment or as traditional natural medicines. The important medicinal properties of B. rotunda majorly derived from flavonoids which are highly sought as pharmaceuticals. In this study, culture conditions for the growth of adventitious roots in liquid suspension cultures were optimized. The highest adventitious root production was achieved when cultured with initial inoculum density of 1.5 g and pH value at 5.8 after five weeks of culture. HPLC analysis discovered that production of valuable flavonoid compounds (pinostrobin, cardamonin and panduratin A) was significantly higher when the adventitious roots were cultured with initial inoculum density of 1.5 g whereas the initial pH medium did not significantly affect flavonoid production.

Fusarium Consortium Populations Associated with Asparagus Crop in Spain and Their Role on Field Decline Syndrome

Asparagus Decline Syndrome (ADS) is one of the main phytosanitary problems of asparagus crop worldwide. Diseased plants and soil samples from 41 fields from three main production areas of Spain were surveyed. Eight Fusarium species belonging to seven species complexes were identified in soils: F. oxysporum, F. proliferatum, F. redolens, F. solanisensu stricto, F. equiseti, F. culmorum, F. compactum and F. acuminatum. Fusarium oxysporum was the most prevalent species. Statistical correlation (R2 = 88%) was established between F. oxysporum inoculum density and the average temperature of the warmest month. A relationship was also established between three crop factors (average temperature, crop age and F. oxysporum inoculum density) and field disease indices. Significant differences were observed between the distribution of F. oxysporum propagules in white and green asparagus fields. Thirteen Fusarium species belonging to seven species complexes were identified from roots of diseased plants, being F. oxysporum the most prevalent. F. proliferatum, F. oxysporum and F. redolens showed pathogenicity to asparagus and were the main species associated to ADS. Fusarium oxysporum was the species with the highest genetic diversity displaying 14 sequence-based haplotypes with no geographic differentiation. This work contributes to understanding the Fusarium complex associated to ADS for developing accurate integrated disease management strategies.

Pseudomonas putida as a potential biocontrol agent against Salmonella Java biofilm formation in the drinking water system of broiler houses

Background Environmental biofilms can induce attachment and protection of other microorganisms including pathogens, but can also prevent them from invasion and colonization. This opens the possibility for so-called biocontrol strategies, wherein microorganisms are applied to control the presence of other microbes. The potential for both positive and negative interactions between microbes, however, raises the need for in depth characterization of the sociobiology of candidate biocontrol agents (BCAs). The inside of the drinking water system (DWS) of broiler houses is an interesting niche to apply BCAs, because contamination of these systems with pathogens plays an important role in the infection of broiler chickens and consequently humans. In this study, Pseudomonas putida, which is part of the natural microbiota in the DWS of broiler houses, was evaluated as BCA against the broiler pathogen Salmonella Java. Results To study the interaction between these species, an in vitro model was developed simulating biofilm formation in the drinking water system of broilers. Dual-species biofilms of P. putida strains P1, P2, and P3 with S. Java were characterized by competitive interactions, independent of P. putida strain, S. Java inoculum density and application order. When equal inocula of S. Java and P. putida strains P1 or P3 were simultaneously applied, the interaction was characterized by mutual inhibition, whereas P. putida strain P2 showed an exploitation of S. Java. Lowering the inoculum density of S. Java changed the interaction with P. putida strain P3 also into an exploitation of S. Java. A further increase in S. Java inhibition was established by P. putida strain P3 forming a mature biofilm before applying S. Java. Conclusions This study provides the first results showing the potential of P. putida as BCA against S. Java in the broiler environment. Future work should include more complex microbial communities residing in the DWS, additional Salmonella strains as well as chemicals typically used to clean and disinfect the system.

Pseudomonas putida as a potential biocontrol agent against Salmonella Java biofilm formation in the drinking water system of broiler houses

Background: Environmental biofilms can induce attachment and protection of other microorganisms including pathogens, but can also prevent them from invasion and colonization. This opens the possibility for so-called biocontrol strategies, wherein microorganisms are applied to control the presence of other microbes. The potential for both positive and negative interactions between microbes, however, raises the need for in depth characterization of the sociobiology of candidate biocontrol agents (BCAs). The inside of the drinking water system (DWS) of broiler houses is an interesting niche to apply BCAs, because contamination of these systems with pathogens plays an important role in the infection of broiler chickens and consequently humans. In this study, Pseudomonas putida, which is part of the natural microbiota in the DWS of broiler houses, was evaluated as BCA against the broiler pathogen Salmonella Java.Results: To study the interaction between these species, an in vitro model was developed simulating biofilm formation in the drinking water system of broilers. Dual-species biofilms of P. putida strains P1, P2, and P3 with S. Java were characterized by competitive interactions, independent of P. putida strain, S. Java inoculum density and application order. When equal inocula of S. Java and P. putida strains P1 or P3 were simultaneously applied, the interaction was characterized by mutual inhibition, whereas P. putida strain P2 showed an exploitation of S. Java. Lowering the inoculum density of S. Java changed the interaction with P. putida strain P3 also into an exploitation of S. Java. A further increase in S. Java inhibition was established by P. putida strain P3 forming a mature biofilm before applying S. Java.Conclusions: This study provides the first results showing the potential of P. putida as BCA against S. Java in the broiler environment. Future work should include more complex microbial communities residing in the DWS, additional Salmonella strains as well as chemicals typically used to clean and disinfect the system.

Effects of Inoculum Density and Cultivar Susceptibility on Rhizoctonia Damping-off and Crown and Root Rot in Sugar Beet

Damping-off and crown and root rot of sugar beet caused by Rhizoctonia solani AG 2-2 are important soilborne diseases in Minnesota and North Dakota. Management involves an integrated approach including crop rotation, use of resistant cultivars, and timely fungicide application. Our objectives were to evaluate the role of inoculum density and cultivar susceptibility on onset and development of Rhizoctonia diseases and on yield and quality in sugar beet. Three cultivars varying in susceptibility were sown in field plots inoculated with 0, 20, 40, or 60 kg ha-1 R. solani AG 2-2 IIIB infested barley during 2013 and 2015. In both years, there was a significant linear effect of inoculum density with decreasing area under the stand establishment curve (AUSEC), root yield, and sucrose quality as inoculum density increased. Cultivar susceptibility significantly affected AUSEC, and sucrose quality in both years and root yield in 2013. In both years, there was an inoculum density by cultivar interaction on disease ratings, with the partially resistant cultivar resulting in lower ratings than moderate and susceptible cultivars, especially as inoculum density increased. Results have implications for cultivar selection and use and timing of postemergence fungicide application based on field history of inoculum pressure.