Geographical variations, prevalence, and molecular dynamics of fastidious phloem-limited pathogens infecting sugar beet across Central Europe.

In Europe, two fastidious phloem-limited pathogens, 'Candidatus Phytoplasma solani' (16SrXII-A) and 'Candidatus Arsenophonus phytopathogenicus', are associated with rubbery taproot disease (RTD) and syndrome basses richesses (SBR) of sugar beet, respectively. Both diseases can significantly reduce yield, especially when accompanied by root rot fungi. This study investigates the presence, geographic distribution and genetic traits of fastidious pathogens and the accompanying fungus, Macrophomina phaseolina, found on sugar beet across four geographically separated plains spanning seven countries in Central Europe. The survey revealed variable incidences of symptoms linked to these fastidious pathogens in the Pannonian and Wallachian Plains, sporadic occurrence in the North European Plain, and no symptomatic sugar beet in the Bohemian Plain. Molecular analyses unveiled the occurrence of both 'Ca. P. solani' and 'Ca. A. phytopathogenicus' throughout Central Europe, with a predominance of the phytoplasma. These fastidious pathogens were detected in all six countries surveyed within the Pannonian and Wallachian Plains, with only a limited presence of various phytoplasmas was found in the North European Plain, while no fastidious pathogens were detected in Bohemia, aligning with observed symptoms. While 16S rDNA sequences of 'Ca. P. solani' remained highly conserved, multi-locus characterization of two more variable loci (tuf and stamp) unveiled distinct variability patterns across the plains. Notably, the surprising lack of variability of tuf and stamp loci within Central Europe, particularly the Pannonian Plain, contrasted their high variability in Eastern and Western Europe, corresponding to epidemic and sporadic occurrence, respectively. The current study provides valuable insights into the genetic dynamics of 'Ca. P. solani' in Central Europe, and novel findings of the presence of 'Ca. A. phytopathogenicus' in five countries (Slovakia, Czech Republic, Austria, Serbia, and Romania) and M. phaseolina in sugar beet in Slovakia. These findings emphasize the need for further investigation of vector-pathogen(s)-plant host interactions and ecological drivers of disease outbreaks.

Limited variation in microbial communities across populations of Macrosteles leafhoppers (Hemiptera: Cicadellidae).

Microbial symbionts play crucial roles in insect biology, yet their diversity, distribution, and temporal dynamics across host populations remain poorly understood. In this study, we investigated the spatio-temporal distribution of bacterial symbionts within the widely distributed and economically significant leafhopper genus Macrosteles, with a focus on Macrosteles laevis. Using host and symbiont marker gene amplicon sequencing, we explored the intricate relationships between these insects and their microbial partners. Our analysis of the cytochrome oxidase subunit I (COI) gene data revealed several intriguing findings. First, there was no strong genetic differentiation across M. laevis populations, suggesting gene flow among them. Second, we observed significant levels of heteroplasmy, indicating the presence of multiple mitochondrial haplotypes within individuals. Third, parasitoid infections were prevalent, highlighting the complex ecological interactions involving leafhoppers. The 16S rRNA data confirmed the universal presence of ancient nutritional endosymbionts-Sulcia and Nasuia-in M. laevis. Additionally, we found a high prevalence of Arsenophonus, another common symbiont. Interestingly, unlike most previously studied species, M. laevis exhibited only occasional cases of infection with known facultative endosymbionts and other bacteria. Notably, there was no significant variation in symbiont prevalence across different populations or among sampling years within the same population. Comparatively, facultative endosymbionts such as Rickettsia, Wolbachia, Cardinium and Lariskella were more common in other Macrosteles species. These findings underscore the importance of considering both host and symbiont dynamics when studying microbial associations. By simultaneously characterizing host and symbiont marker gene amplicons in large insect collections, we gain valuable insights into the intricate interplay between insects and their microbial partners. Understanding these dynamics contributes to our broader comprehension of host-microbe interactions in natural ecosystems.

Tomato and Jute Mallow are Two New Hosts of Papaya Bunchy Top Phytoplasma, a 16SrXII-O Subgroup Strain in Nigeria.

Nigerian papaya bunchy top (NGPBT) phytoplasma was first identified in diseased papaya plants growing in Ibadan, Oyo State, Nigeria (Kazeem et al. 2021). The NGPBT phytoplasma is a 'Candidatus Phytoplasma convolvuli'-related strain and represents a subgroup lineage, 16SrXII-O (the accession number of the reference strain is MW530522, Kazeem et al. 2021). The present communication reports that NGPBT phytoplasma can also infect tomato (Solanum lycopersicum) and jute mallow (Corchorus olitorius). Since May 2020, tomato and jute mallow grwn in Ibadan have been observed to develop yellowing, little leaf, and stunting symptoms (Fig. 1). Because the symptomatic plants occurred in the region approximately 1 km adjacent to where the NGPBT disease was reported, and the symptoms of infected plants resembled those of phytoplasma infection, molecular diagnostic assays for phytoplasma detection were deployed. Total DNAs were extracted from symptomatic plants, including four tomato plants and three jute mallows, as well as from asymptomatic two tomato and two jute mallow plants. The DNA samples were subjected to semi-nested PCR using phytoplasma 16S rRNA gene-specific primers P1A and P7A, followed by P1A and 16S-SR (Lee et al. 2004). An amplicon of 1.5 kb was obtained from each of the symptomatic plants, while no amplicon resulted from DNA samples of asymptomatic plants or negative controls without DNA templates (water and PCR reagents only). PCR products were cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA), and three clones were chosen for each sample for Sanger sequencing (Psomagen Inc., Rockville, MD, USA). The nearly full-length 16S rRNA gene sequences (1.53kb) derived from tomato (OP123558) and jute mallow (OP123559) samples were identical. Based on the iPhyClassifier phytoplasma classification web tool (Zhao et al. 2009) and the BLAST search against the NCBI nucleotide database, these phytoplasma strains showed 100% sequence identity in 16S rRNA gene with the NGPBT phytoplasma (16SrXII-O, MW530522). Moreover, two additional genetic loci including ribosomal protein genes rplV-rpsC, and rplO-secY-adk were also amplified by nested PCR or semi-nested PCR with specific primers rpStolF/rpStolR followed by rpStolF2/rpStolR (Martini et al. 2007), and SecYF1a (Xll)/MapR-703-a, followed by SecYF2a (Xll)/MapR-703-a (Lee et al. 2010). Gene fragments of rplV-rpsC (1238bp) and rplO-secY-adk (2064bp) were amplified from DNAs of diseased papaya, tomato, and jute mallow plants. The obtained sequences were deposited into GenBank, respectively: rplV-rpsC (OP123560, OP123562, and OP123563) and rplO-secY-adk (OP123565, OP123567, and OP123568). Multilocus sequence analysis (MLSA) indicated that the sequences of phytoplasmas amplified from three different plant hosts were also identical in rp, secY, and adk genes. The MLSA results demonstrate that tomato and jute mallow are two new hosts of NGPBT phytoplasmas. This also marks the first time that phytoplasma diseases are associated with tomato and jute mallow in Nigeria, as prior to this study, phytoplasma diseases were only reported in coconut palm and papaya in the country (Osagie et al. 2016; Kazeem et al. 2021). Results from the present study suggest that insect vector(s) for the transmission of the NGPBT phytoplasma are present in the region. Since both tomato and jute mallow are important vegetable crops in Nigeria, timely dissemination of emerging disease information is needed to alert growers and extension personnel in the region. In addition, ongoing incidence, and prevalence surveys of NGPBT disease indicate that more infected papaya and tomato plants have been observed in the region than in previous years. A better understanding of the NGPBT phytoplasma disease epidemiology will help devise strategies to control the diseases associated with the NGPBT phytoplasma.

'Candidatus Phytoplasma asteris' subgroups display distinct disease progression dynamics during the carrot growing season.

Aster Yellows phytoplasma (AYp; 'Candidatus Phytoplasma asteris') is an obligate bacterial pathogen that is the causative agent of multiple diseases in herbaceous plants. While this phytoplasma has been examined in depth for its disease characteristics, knowledge about the spatial and temporal dynamics of pathogen spread is lacking. The phytoplasma is found in plant's phloem and is vectored by leafhoppers (Cicadellidae: Hemiptera), including the aster leafhopper, Macrosteles quadrilineatus Forbes. The aster leafhopper is a migratory insect pest that overwinters in the southern United States, and historical data suggest these insects migrate from southern overwintering locations to northern latitudes annually, transmitting and driving phytoplasma infection rates as they migrate. A more in-depth understanding of the spatial, temporal and genetic determinants of Aster Yellows disease progress will lead to better integrated pest management strategies for Aster Yellows disease control. Carrot, Daucus carota L., plots were established at two planting densities in central Wisconsin and monitored during the 2018 growing season for Aster Yellows disease progression. Symptomatic carrots were sampled and assayed for the presence of the Aster Yellows phytoplasma. Aster Yellows disease progression was determined to be significantly associated with calendar date, crop density, location within the field, and phytoplasma subgroup.

Complete Genome Sequence of "Candidatus Phytoplasma asteris" RP166, a Plant Pathogen Associated with Rapeseed Phyllody Disease in Poland.

The complete genome sequence of "Candidatus Phytoplasma asteris" RP166, which consists of one 829,546-bp circular chromosome, is presented in this work. This bacterium is associated with rapeseed phyllody disease in Poland and belongs to the 16SrI-B (i.e., aster yellows) group.

Genetic Variation Among Geographically Disparate Isolates of Aster Yellows Phytoplasma in the Contiguous United States.

Aster Yellows phytoplasma (AYp; Candidatus Phytoplasma asteris) is associated with diseases of herbaceous plants, including ornamentals and important commercial vegetable and grain crops. The aster leafhopper (ALH; Macrosteles quadrilineatus Forbes) is the predominant vector of these bacteria, though other leafhopper species can acquire and transmit AYp. Potentially inoculative leafhoppers are reported to overwinter in the southern United States and migrate to northern latitudes in the spring. Examining the genetic similarities and differences in AYp associated with southern and northern populations of ALH may provide insight into the role that migrating ALH play in AYp disease development. To investigate similarities among geographically distinct populations of ALH and characterize the variation in AYp associated within these populations, we identified genetic variations in subgroup designation and the relative proportions of secreted AY-WB proteins from field-collected populations of AYp isolated from ALH from select locations in the southern (Arkansas, Kansas, Oklahoma, and Texas) and the northern United States (Wisconsin) in 2016, 2017, and 2018. Isolated phytoplasma were tested for variation of AYp genotypes, numbers of potentially inoculative (AYp-positive) ALH, and presence of specific AYp virulence (effector) genes. Geographically distinct populations of ALH collected in northern and southern regions were similar in CO1 genotype but carried different proportions of AYp genotypes. While similar AYp strains were detected in geographically distinct locations, the proportion of each genotype varied over time.

Two high-copy plasmids found in plants associated with strains of "Candidatus Phytoplasma asteris".

Complete sequences for two plasmids associated with two strains of "Candidatus Phytoplasma asteris" have been obtained. The plasmid named pPARG1 was found in Rehmannia glutinosa L. associated with phytoplasma classified in the 16Sr I-C subgroup. Plasmid pPABN1 was from phytoplasma associated with infected winter oilseed rape and classified in the 16Sr I-B subgroup. The plasmids pPARG1 (4371 nt) and pPABN1 (3529 nt) have high A+T content of about 75%, similar to that of phytoplasma genomes. Six and five open reading frames longer than 100 amino acids and organized on the same strand were recognized on pPARG1 and pPABN1, respectively. A segment about 1.6 kbp long sharing high sequence identity with the Onion yellows phytoplasma genome was found in pPABN1.