Publications

4465

Abstract Many biotechnological applications deal with nitrification, one of the main steps of the global nitrogen cycle. The biological oxidation of ammonia to nitrite and further to nitrate is critical to avoid environmental damage and its functioning has to be retained even under adverse conditions. Bacteria performing the second reaction, oxidation of nitrite to nitrate, are fastidious microorganisms that are highly sensitive against disturbances. One important finding with relevance for nitrogen removal systems was the discovery of the mainly cold-adapted Cand. Nitrotoga, whose activity seems to be essential for the recovery of nitrite oxidation in wastewater treatment plants at low temperatures, e.g., during cold seasons. Several new strains of this genus have been recently described and ecophysiologically characterized including genome analyses. With increasing diversity, also mesophilic Cand. Nitrotoga representatives have been detected in activated sludge. This review summarizes the natural distribution and driving forces defining niche separation in artificial nitrification systems. Further critical aspects for the competition with Nitrospira and Nitrobacter are discussed. Knowledge about the physiological capacities and limits of Cand. Nitrotoga can help to define physico-chemical parameters for example in reactor systems that need to be run at low temperatures. Key points • Characterization of the psychrotolerant nitrite oxidizer Cand. Nitrotoga • Comparison of the physiological features of Cand. Nitrotoga with those of other NOB • Identification of beneficial environmental/operational parameters for proliferation

The phloem-limited ‘Candidatus Liberibacter asiaticus’ (Las) causes huanglongbing, a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in the greenhouse, and the impacts of temperature on plant colonization in growth-chamber experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by quantitative PCR (qPCR) or symptoms at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, maintained at from 8 to 20°C, from 18 to 30°C, or from 24 to 38°C daily range, and assessed by qPCR of samples taken from noninoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to analysis of variance and binomial generalized linear model and logistic regression analyses. Probabilities of detecting Las in greenhouse plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day−1 average speed of movement. In growth chambers, the temperature impacted plant colonization by Las, new shoot emission, and symptom expression. After a 7-month exposure time, Las was absent in all new shoots in the cooler environment (average three per plant), and present in 70% at the milder environment (six shoots, severe symptoms) and 25% in the warmer environment (eight shoots, no visible symptoms). Temperature of 25.7°C was the optimum condition for plant colonization. This explains the higher impact and incidence of huanglongbing disease during the winter months or regions of milder climates in Brazil.

Tomato is one of the most economically important crops in Republic of Moldova. However, it is affected by a number of pathogens. One of the wide spread diseases is stolbur caused by the infection agent ‘Candidatus Phytoplasma solani’. Accurate diagnostics of the disease at an early stage is essential for successful control of the disease. In this work, we describe detection and quantification of ‘Candida-tus Phytoplasma solani’ in tomato by real-time PCR, as well as suitability of the method for assessing resistance of different tomato varieties to Phytoplasma infection.

Pythium terrestris, Pythium spinosum, and ‘Candidatus Pythium huanghuaiense’ are closely related species and important pathogens of soybean that cause root rot. However, the sequences of commonly used molecular markers, such as rDNA internal transcribed spacer 2 and cytochrome oxidase 1 gene, are similar among these species, making it difficult to design species-specific primers for loop-mediated isothermal amplification (LAMP) assays. The genome sequences of these species are also currently unavailable. Based on a comparative genomic analysis and de novo RNA-sequencing transcript assemblies, we identified and cloned the sequences of the M90 gene, a conserved but highly polymorphic single-copy gene encoding a Puf family RNA-binding protein among oomycetes. After primer design and screening, three LAMP assays were developed that specifically amplified the targeted DNA sequences in P. terrestris and P. spinosum at 62°C for 70 min and in ‘Ca. Pythium huanghuaiense’ at 62°C for 60 min. After adding SYBR Green I, a positive yellow-green color (under natural light) or intense green fluorescence (under ultraviolet light) was observed by the naked eye only in the presence of the target species. The minimum concentration of target DNA detected in all three LAMP assays was 100 pg·μl−1. The assays also successfully detected the target Pythium spp. with high accuracy and sensitivity from inoculated soybean seedlings and soils collected from soybean fields. This study provides a method for identification and cloning of candidate detection targets without a reference genome sequence and identified M90 as a novel specific target for molecular detection of three Pythium species causing soybean root rot.