Publications

4504

Summary Huanglongbing (HLB) is currently considered the most destructive disease of citrus worldwide. In the major citrus‐growing areas in Asia and the US, the major causal agent of HLB is the bacterial pathogen Candidatus Liberibacter asiaticus (CLas). CLas is vectored by the Asian citrus psyllid, Diaphorina citri , in a persistent propagative manner. CLas cannot be cultured in vitro because of its unclear growth factors, leading to uncertainty in the infection mechanism of CLas at the cellular level in citrus and in D. citri . To characterize the detailed infection of CLas in the host and vector, the incidence of HLB was first investigated in citrus‐growing fields in Fujian Province, China. It was found that the positive association of the level of CLas infection in the leaves correlated with the symptoms. Then antibodies against peptides of the outer membrane protein (OMP) of CLas were prepared and tested. The antibodies OMP‐225, OMP‐333 and OMP724 showed specificity to citrus plants in western blot analyses, whereas the antibodies OMP‐47 and OMP‐225 displayed specificity to the D. citri vector. The application of OMP‐225 in the immunofluorescence assay indicated that CLas was located in and distributed throughout the phloem sieve cells of the leaf midribs and axile placenta of the fruit. CLas also infected the epithelial cells and visceral muscles of the alimentary canal of D. citri . The application of OMP‐333 in immunoelectron microscopy indicated the round or oval CLas in the sieve cells of leaf midribs and axile placenta of fruit as well as in the epithelial cells and reticular tissue of D. citri alimentary canal. These results provide a reliable means for HLB detection, and enlighten a strategy via neutralizing OMP to control HLB. These findings also provide insight for the further investigation on CLas infection and pathogenesis, as well as CLas–vector interaction.

Changes in physiological and biochemical patterns in lucerne plants caused by the presence of ‘Candidatus Phytoplasma australasia’, which is one of the significant pathogens causing yield losses in lucerne plants, were investigated. Significant differences were evident in total chlorophyll, chlorophyll a, chlorophyll b, and protein amounts between ‘Ca. Phytoplasma australasia’-positive and negative lucerne plants. Stress-related metabolites such as phenol, malondialdehyde, and proline accumulations in ‘Ca. Phytoplasma australasia’-positive plants were remarkably higher than those of phytoplasma-negative plants. As a response to disease attack, phytoplasma-positive plants exhibited higher antioxidant enzymes (peroxidase and catalase) and non-enzymatic metabolite responses such as jasmonic and salicylic acids. We state that partial disease responses were revealed for the first time to breed resistant lucerne lines infected by ‘Ca. Phytoplasma australasia’.

AbstractPeach trees showing witches’‐broom disease symptoms in the northwest of Iran were sampled for phytoplasma detection. PCR assays and Sanger sequence analyses indicated that ʻCandidatus Phytoplasma phoeniciumʼ was associated with peach witchesʼ‐broom disease. Virtual RFLP analyses of the 16S rRNA gene indicated that ʻCa. Phytoplasma phoeniciumʼ strain, which was prevalent in the northwest of Iran belonged to 16SrIX‐C subgroup. For the genomic characterization of Iranian ʻCa. Phytoplasma phoeniciumʼ strain, total DNA extracted from the infected peach trees was subjected to Illumina next‐generation sequencing. The NGS sequencing resulted in 41157647 read pairs of raw data. The raw reads length was 150 bp and the insert size was 350 bp. Assembly and genes clustering finally resulted in 750 contigs with a total size of 228345 bp. The GC content of sequence was 33.2%, N50 value was 259 and L50 value was 256. According to NCBI‐ PGAP annotation the genome of Prunus persica phytoplasma PP2 contained 410 genes including 377 CDSs with protein, 10 rRNAs, nine tRNAs and 14 pseudogenes.

AbstractThe Yessentukskoye deposit of Caucasian mineral waters contains balneologically valuable drinking mineral water, which is extracted from the Upper Cretaceous 1 km subsurface aquifer and is almost unexplored by microbiologists. We have sampled this water via continuously operating production wells, characterized the phylogenetic diversity of its microbial community, and obtained enrichments of thermophilic iron reducers from the source aquifer. From the enrichments, a novel anaerobic thermophilic bacterium, reducing Fe(III) in the mineral ferrihydrite with acetate as the electron donor, was isolated into a pure culture. The novel isolate, designated as strain Es71-Z0220Tbelonging toDeferribacteralesorder, is thermophilic, neutrophilic, halotolerant, motile vibrio. It utilizes synthesized ferrihydrite, fumarate, nitrate or elemental sulfur as the electron acceptors with organic acids as the electron donors. The strain is incapable of soluble Fe(III) complexes reduction and fermentative growth. The draft genome assembly of strain Es71-Z0220Tresulted in 65 contigs with a total size of ca. 2.3 Mb. On the basis of whole-genome phylogenetic reconstruction and physiological characterization, the novel isolate was considered to represent a novel family, genus and species for which the nameDeferrivibrio essentukiensisgen. nov., sp. nov. is proposed. Genome analysis revealed key determinants of anaerobic respiration and carbon substrate utilization pathways in the organism with peculiarities related to putative Fe(III)-reducing electron transfer chain. Considering the revealed metabolic features ofDeferrivibrio essentukiensis, the involvement of the organism in its subsurface environment in biogeochemical by carbon cycling by coupling the organic matter oxidation with Fe(III) minerals reduction is discussed.

Abstract‘Candidatus Phytoplasma ulmi’, associated with elm yellows, shoot proliferation and dieback of elm (Ulmus) species trees was reported in United States of America and in many European countries. Until now its presence in elm trees in Poland has not been detected. In 2017–2018, during visual inspection of elm trees grown in four areas of southern Silesia Province leaf yellowing, shoot proliferation, phloem necrosis and dieback of branches were observed on European field elm trees grown in nature reserve close to the border with the Czech Republic. Phloem tissue from shoots collected from six symptomatic and twenty asymptomatic trees was used for extraction nucleic acids. Nested PCR with primer pairs amplifying 16Sr RNA sequences of phytoplasmas resulted in specific products for samples from six symptomatic trees however the restriction fragment length polymorphism (RFLP) and phylogenetic analyses of the sequenced products did not allow to clearly classify newly detected isolates within the elm yellows group (16SrV). The isolates found in Poland clustered on the boundary of the 16SrV‐A and 16SrV‐C ribosomal subgroups. Sequence analyses of less conservative genes rpl22‐ rps3 and secY confirmed the affiliation of the phytoplasma isolates found in Poland to ‘Candidatus Phytoplasma ulmi’; however, the significant nucleotide changes in signature sequences for all three genes were shown compared with EY1T reference strain. Surprisingly, the newly detected isolates shared a common origin and were phylogenetically closer to EY1‐SRB and EY10‐SRB strains from Serbia as well as NK16 strain from Croatia strains than to the EYCZ1 strain from the Czech Republic. This is the first report on detection and molecular characterization of ‘Candidatus Phytoplasma ulmi’ in Poland based on sequence analysis of three genetic locus.

Abstract Background The tomato psyllid, Bactericera cockerelli Šulc (Hemiptera: Triozidae), is a pest of solanaceous crops such as tomato (Solanum lycopersicum L.) in the U.S. and vectors the disease-causing pathogen ‘Candidatus Liberibacter solanacearum’ (or Lso). Disease symptom severity is dependent on Lso haplotype: tomato plants infected with Lso haplotype B experience more severe symptoms and higher mortality compared to plants infected with Lso haplotype A. By characterizing the molecular differences in the tomato plant’s responses to Lso haplotypes, the key components of LsoB virulence can be identified and, thus, targeted for disease mitigation strategies. Results To characterize the tomato plant genes putatively involved in the differential immune responses to Lso haplotypes A and B, RNA was extracted from tomato ‘Moneymaker’ leaves 3 weeks after psyllid infestation. Gene expression levels were compared between uninfected tomato plants (i.e., controls and plants infested with Lso-free psyllids) and infected plants (i.e., plants infested with psyllids infected with either Lso haplotype A or Lso haplotype B). Furthermore, expression levels were compared between plants infected with Lso haplotype A and plants infected with Lso haplotype B. A whole transcriptome analysis identified 578 differentially expressed genes (DEGs) between uninfected and infected plants as well as 451 DEGs between LsoA- and LsoB-infected plants. These DEGs were primarily associated with plant defense against abiotic and biotic stressors, growth/development, plant primary metabolism, transport and signaling, and transcription/translation. These gene expression changes suggested that tomato plants traded off plant growth and homeostasis for improved defense against pathogens, especially when infected with LsoB. Consistent with these results, tomato plant growth experiments determined that LsoB-infected plants were significantly stunted and had impaired negative geotropism. However, it appeared that the defense responses mounted by tomatoes were insufficient for overcoming the disease symptoms and mortality caused by LsoB infection, while these defenses could compensate for LsoA infection. Conclusion The transcriptomic analysis and growth experiments demonstrated that Lso-infected tomato plants underwent gene expression changes related to abiotic and biotic stressors, impaired growth/development, impaired plant primary metabolism, impaired transport and signaling transduction, and impaired transcription/translation. Furthermore, the transcriptomic analysis also showed that LsoB-infected plants, relative to LsoA-infected, experienced more severe stunting, had improved responses to some stressors and impaired responses to others, had poorer transport and signaling transduction, and had impaired carbohydrate synthesis and photosynthesis.

Grapevine “bois noir”, related to the presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), represents a serious threat in several vine-growing areas worldwide. In surveys conducted over two years, mild and/or moderate symptoms and lower pathogen titer were mainly associated with ‘Ca. P. solani’ strains harboring a secY gene sequence variant (secY52), whereas severe symptoms and higher titer were mainly observed in grapevines infected by phytoplasma strains carrying any one of another four variants. A comparison of amino acid sequences of the protein SecY of ‘Ca. P. solani’ strains revealed the presence of conservative and semi-conservative substitutions. The deduced three-dimensional (3D) protein analysis unveiled that one semi-conservative substitution identified in the sequence variant secY52 is responsible for a structural disordered region that probably confers a flexibility for binding to distinct molecular complexes. In fact, the other analyzed variants show an organized structure and the 3D in silico prediction allowed the identification of β-sheets. Thus, differences in symptom severity and pathogen concentration observed in grapevines infected by ‘Ca. P. solani’ strains carrying distinct secY gene sequence variants suggest a possible relationship between SecY protein structure and phytoplasma strain virulence.

Diaphorina citri is a serious insect pest of citrus and an insect vector of Candidatus Liberibacter asiaticus (CLas) that causes Huanglongbing disease (citrus greening). In this study, we investigated the effect of the CLas pathogen on the life history parameters of D. citri at different temperature regimes. Our results demonstrated that the survival rate of first to fifth instar CLas-positive and CLas-negative D. citri fluctuate with the change in temperature over the range of 16–35 °C. Meanwhile, the mean developmental time (52.5 d) (d = day(s)) and adult longevity (5.2 d) of the CLas-positive psyllids was longer as compared to CLas-negative psyllids mean developmental time (32.81 d) and adult longevity (3.50 d) at the low- and high-temperature regimes (16 and 35 °C). However, at high temperature regimes, the significant effect of CLas-bacteria on D. citri fecundity was higher than the corresponding non-significant effect on their survivorship when compared to non-vectored psyllids. These results indicate a long-term, stable evolutionary relationship among vector-pathogen and climate change.