Plant Disease

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 potato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) is a pest of solanaceous crops (order Solanales), including potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.). Feeding by high populations of nymphs causes psyllid yellows while adults and nymphs are vectors of the plant pathogen ‘Candidatus Liberibacter solanacearum’. Foliar symptoms that were consistent with either ‘Ca. L. solanacearum’ infection or psyllid yellows were observed in 2019 on tomatillo (Physalis ixocarpa Brot.; family Solanaceae) grown within an experimental plot located near Saltillo, Mexico. This study had three primary objectives: 9i) determine whether the foliar symptoms observed on tomatillo were associated with ‘Ca. L. solanacearum’ infection, (ii) identify the haplotypes of ‘Ca. L. solanacearum’ and potato psyllids present in the symptomatic plot, and (iii) use gut content analysis to infer the plant sources of ‘Ca. L. solanacearum’-infected psyllids. Results confirmed that 71% of symptomatic plants and 71% of psyllids collected from the plants were infected with ‘Ca. L. solanacearum’. The detection of ‘Ca. L. solanacearum’ in plants and psyllids and the lack of nymphal populations associated with psyllid yellows strongly suggests that the observed foliar symptoms were caused by ‘Ca. L. solanacearum’ infection. All infected plants and insects harbored the more virulent ‘Ca. L. solanacearum’ haplotype B but one psyllid was also coinfected with haplotype A. The potato psyllids were predominantly of the central haplotype but one psyllid was identified as the western haplotype. Molecular gut content analysis of psyllids confirmed the movement of psyllids between noncrop habitats and tomatillo and indicated that ‘Ca. L. solanacearum’ infection of psyllids was associated with increased plant diversity in their diet.

‘Candidatus Liberibacter asiaticus’ is an insect-transmitted, phloem-restricted α-proteobacterium associated with huanglongbing. Here, we provide the whole genome sequence of ‘Ca. L. asiaticus’ strain ReuSP1 from its insect vector Diaphorina citri (Hemiptera: Liviidae) collected in La Réunion. The genome is composed of 1,230,064 bp and has a 36.5% G+C content. This study reports the first ‘Ca. L. asiaticus’ genome sequence from La Réunion, which will add to ‘Ca. L. asiaticus’ genome resources and help elucidate our understanding of the introduction pathway into La Réunion.

‘Candidatus Liberibacter asiaticus,’ an uncultured α-proteobacterium, is associated with citrus huanglongbing (HLB, yellow shoot disease), a destructive disease threatening citrus production worldwide. Here, we reported the draft genome sequence of ‘Ca. L. asiaticus’ strain Myan16 from an HLB-affected lime tree in Myitkyina, Kachin State, Myanmar. The strain Myan16 genome is 1,229,102 bp with an average G+C content of 36.4%, along with a circular prophage: P-Myan16-2 (36,303 bp, type 2). This is the first genome sequence of a ‘Ca. L. asiaticus’ strain from Myanmar, which will enrich the current ‘Ca. L. asiaticus’ genome sequence database and facilitate HLB epidemiology research in Asia and world.

Rubbery taproot disease (RTD) of sugar beet was observed in Serbia for the first time in the 1960s. The disease was already described in neighboring Bulgaria and Romania at the time but it was associated with abiotic factors. In this study on RTD of sugar beet in its main growing area of Serbia, we provide evidence of the association between ‘Candidatus Phytoplasma solani’ (stolbur phytoplasma) infection and the occurrence of typical RTD symptomatology. ‘Ca. P. solani’ was identified by PCR and the sequence analyses of 16S ribosomal RNA, tuf, secY, and stamp genes. In contrast, the causative agent of the syndrome “basses richesses” of sugar beet—namely, ‘Ca. Arsenophonus phytopathogenicus’—was not detected. Sequence analysis of the stolbur strain’s tuf gene confirmed a previously reported and a new, distinct tuf stolbur genotype (named ‘tuf d’) that is prevalent in sugar beet. The sequence signatures of the tuf gene as well as the one of stamp both correlate with the epidemiological cycle and reservoir plant host. This study provides knowledge that, for the first time, enables the differentiation of stolbur strains associated with RTD of sugar beet from closely related strains, thereby providing necessary information for further epidemiological work seeking to identify insect vectors and reservoir plant hosts. The results of this study indicate that there are differences in hybrid susceptibility. Clarifying the etiology of RTD as a long-known and economically important disease is certainly the first step toward disease management in Serbia and neighboring countries.

‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable, phloem-limited, insect-transmitted bacterium associated with the Asiatic form of huanglongbing (HLB), the most destructive citrus disease. In Asia and the Americas, it is transmitted by the Asian citrus psyllid (Diaphorina citri Kuwavama). Despite considerable research, little is known about the processes involved in plant infection and colonization by Las. This study was conducted to determine whether the basal portion (below girdling) of the plant is an important route for Las to move laterally from a point of inoculation on a branch to pathogen-free branches elsewhere in the canopy, and to quantify the influence of actively growing tissues on vertical upward (acropetally) or downward (basipetally) movement of Las. Nongirdled and fully or partially girdled stems of potted plants of ‘Pera’ sweet orange, graft-inoculated above or below girdling, were sampled in distinct regions and assessed by qPCR, 6 months postinoculation. Las invaded all regions of partially and nongirdled plants but remained restricted to the inoculated regions of fully girdled plants, evidence that in planta bacterium movement is limited to the phloem. In fully girdled plants, starch accumulated above the girdling site, probably because of changes in flow of phloem sap. To study the influence of actively growing tissues, inoculated ‘Valencia’ sweet orange plants were kept intact or were top- or root-pruned to force production of new tissues, and sampled at 15-day intervals. Las migrated rapidly and most predominantly toward newly developing root and leaf tissues. The rapid and predominant movement of Las to newly developed shoots and roots would explain failures of canopy heat treatments and pruning to cure HLB-affected trees, and reinforces the need to protect rapidly growing new shoots from feeding by D. citri in order to minimize transmission and spread of the pathogen by the vector within and between orchards.

‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable phloem-restricted α-proteobacterium associated with huanglongbing (HLB). Here, we provide the genome sequence of CLas strain CoFLP1 from its insect vector Diaphorina citri (Hemiptera: Liviidae) collected in the department of La Guajira, Colombia. The CoFLP1 strain is composed of 1,231,639 bp with G+C 36.5% content. This study reports the first CLas genome sequence from Colombia, which will add to CLas genome resources and help to elucidate our understanding of the introduction pathway of HLB in South America.