Monitoring the Photorhabdus spp. bacterial load in Heterorhabditis bacteriophora dauer juveniles over different storage times and temperatures: A molecular approach.

Biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora can vary in virulence (quality). The influence of their symbiotic bacteria Photorhabdus spp. inside the infective dauer juvenile (DJ) on DJ quality has not received much attention in the past. The presence of the bacteria in the DJ is crucial for its biocontrol potential. This investigation provides a method to quantify the bacterial load inside the DJ based on a qPCR technique. Information from the genome of Photorhabdus laumondii strain DE2 was used to identify single copy genes with no homology to any other bacterial accessions. One gene (hereby named CG2) was selected for primers design and for further qPCR experiments. Cross-amplification tests with P. thracensis and P. kayaii, also symbionts of H. bacteriophora, were positive, whereas no amplicons were produced for P. temperata or Xenorhabdus nematophila. We tested our qPCR system in DJ populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the virulence was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this factor correlated with the reduction of bacterial DNA on the respective DJ population. We observed that stored DJ kept virulent up to 90 days and thereafter the virulence as well as the amount of bacterial DNA drastically decreased. Storage temperature also influenced the bacterial survival. Inside formulated DJ, the loss of bacterial DNA on the DJ population was accelerated under storage temperatures below 7.5 °C, suggesting that reproduction of the bacterial cells takes place when growth temperature is favorable. The role of bacterial survival inside stored DJ can now be adequately addressed using this molecular quality-control technique.

Entomopathogenic nematodes for biological control of Psylliodes chrysocephala (Coleoptera: Chrysomelidae) in oilseed rape.

Winter oilseed rape (Brassica napus) is one of the largest crops in Europe and the cabbage stem flea beetle Psylliodes chrysocephala is one of its major pests. Since the ban of neonicotinoids for seed treatment, farmers apply pyrethroids in autumn to control the cabbage stem flea beetle. Current studies show that the insect develops resistance to this group of chemicals. Biological control with entomopathogenic nematodes (EPNs) represents a possible, environmentally friendly alternative control measure. In the present work, we considered three strategies to control the cabbage stem flea beetle: applying the nematodes against the first larval stage in the soil, against the second and third larval stages inside the plant or against the adult beetles. In laboratory experiments, we found the third larval instar to be the most susceptible stage and the adult beetle the less susceptible one. Steinernema feltiae and the cold active SDT1-IL1 Heterorhabditis bacteriophora strain, with a reduction potential of 89 and 76 %, respectively, proved to be the most virulent EPNs against P. chrysocephala in pot experiments at 15 °C. Moreover, we performed four field trials to test the efficacy of H. bacteriophora and S. feltiae against the larvae. The highest reduction in the field trials was 45% and 39%, obtained with SDT1-IL1 and a mixture of H. bacteriophora and S.feltiae, respectively. The present study provides preliminary information about the potential of EPNs to control P. chrysocephala and represents a start point for the development of a competitive and sustainable alternative to pyrethroids.

Gluconacetobacter diazotrophicus Pal5 Enhances Plant Robustness Status under the Combination of Moderate Drought and Low Nitrogen Stress in Zea mays L.

Plant growth promoting endophytic bacteria, which can fix nitrogen, plays a vital role in plant growth promotion. Previous authors have evaluated the effect of Gluconacetobacter diazotrophicus Pal5 inoculation on plants subjected to different sources of abiotic stress on an individual basis. The present study aimed to appraise the effect of G. diazotrophicus inoculation on the amelioration of the individual and combined effects of drought and nitrogen stress in maize plants (Zea mays L.). A pot experiment was conducted whereby treatments consisted of maize plants cultivated under drought stress, in soil with a low nitrogen concentration and these two stress sources combined, with and without G. diazotrophicus seed inoculation. The inoculated plants showed increased plant biomass, chlorophyll content, plant nitrogen uptake, and water use efficiency. A general increase in copy numbers of G. diazotrophicus, based on 16S rRNA gene quantification, was detected under combined moderate stress, in addition to an increase in the abundance of genes involved in N fixation (nifH). Endophytic colonization of bacteria was negatively affected by severe stress treatments. Overall, G. diazotrophicus Pal5 can be considered as an effective tool to increase maize crop production under drought conditions with low application of nitrogen fertilizer.