Agronomy and Crop Science

The interactions between the phloem-limited pathogen ‘Candidatus Liberibacter solanacearum’ haplotype C and carrot (Daucus carota subsp. sativus) were studied at 4, 5, and 9 weeks postinoculation (wpi), by combining dual RNA-Seq results with data on bacterial colonization and observations of the plant phenotype. In the infected plants, genes involved in jasmonate biosynthesis, salicylate signaling, pathogen-associated molecular pattern- and effector-triggered immunity, and production of pathogenesis-related proteins were up-regulated. At 4 wpi, terpenoid synthesis-related genes were up-regulated, presumably as a response to the psyllid feeding, whereas at 5 and 9 wpi, genes involved in both the terpenoid and flavonoid production were down-regulated and phenylpropanoid genes were up-regulated. Chloroplast-related gene expression was down-regulated, in concordance with the observed yellowing of the infected plant leaves. Both the RNA-Seq data and electron microscopy suggested callose accumulation in the infected phloem vessels, likely to impair the transport of photosynthates, while phloem regeneration was suggested by the formation of new sieve cells and the upregulation of cell wall–related gene expression. The ‘Ca. L. solanacearum’ genes involved in replication, transcription, and translation were expressed at high levels at 4 and 5 wpi, whereas, at 9 wpi, the Flp pilus genes were highly expressed, suggesting adherence and reduced mobility of the bacteria. The ‘Ca. L. solanacearum’ genes encoding ATP and C4-dicarboxylate uptake were differentially expressed between the early and late infection stages, suggesting a change in the dependence on different host-derived energy sources. HPE1 effector and salicylate hydroxylase were expressed, presumably to suppress host cell death and salicylic acid-dependent defenses during the infection. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

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.