Publications

4490

Citrus Huanglongbing (HLB) caused by ‘ Candidatus Liberibacter asiaticus’ (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves—late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

A new disease syndrome of zucchini squash was observed in Southern Italy, in 2018 and again in 2020. Affected plants were severely stunted and leaves were bent downwards, small, stiff, thick, leathery, and had interveinal chloroses. In addition, flowers were virescent and fruits were deformed and often cracked. Disease incidence was 20 and 30% in two different zucchini cultivations in Campania region (Southern Italy). Tomato yellow leaf curl New Delhi virus (ToLCNDV) was detected in eight samples, by loop-mediated isothermal amplification–based (LAMP) kit and by PCR and Sanger sequencing of the AV1 gene. Phytoplasmas were detected in the same samples using nested PCR assays with primer pairs P1/P7 and R16F2n/R16R2. Phytoplasma associations in plant samples were confirmed using specific primers for the multilocus genes SecY, tuf and rp. Sequence comparison of multilocus genes and phylogenetic analyses of the 16S rDNA gene confirmed the association of a phytoplasma strain closely related to ‘Candidiatus Phytoplamsa asteris’. This is the first report of mixed infections of ToLCNDV and a putative ‘Ca. Phytoplamsa asteris’ strain in zucchini, associated with a new Squash-Phytoplasma-Begomovirus (SqPB) disease syndrome.

Abstract Backgroud The genus Mesorhizobium is shown by phylogenomics to be paraphyletic and forms part of a complex that includes the genera Aminobacter, Aquamicrobium, Pseudaminobacter and Tianweitania. The relationships for type strains belong to these genera need to be carefully re-evaluated. Results The relationships of Mesorhizobium complex are evaluated based on phylogenomic analyses and overall genome relatedness indices (OGRIs) of 61 type strains. According to the maximum likelihood phylogenetic tree based on concatenated sequences of 539 core proteins and the tree constructed using the bac120 bacterial marker set from Genome Taxonomy Database, 65 type strains were grouped into 9 clusters. Moreover, 10 subclusters were identified based on the OGRIs including average nucleotide identity (ANI), average amino acid identity (AAI) and core-proteome average amino acid identity (cAAI), with AAI and cAAI showing a clear intra- and inter-(sub)cluster gaps of 77.40–80.91% and 83.98–86.16%, respectively. Combined with the phylogenetic trees and OGRIs, the type strains were reclassified into 15 genera. This list includes five defined genera Mesorhizobium, Aquamicrobium, Pseudaminobacter, Aminobacterand Tianweitania, among which 40/41 Mesorhizobium species and one Aminobacter species are canonical legume microsymbionts. The other nine (sub)clusters are classified as novel genera. Cluster III, comprising symbiotic M. alhagi and M. camelthorni, is classified as Allomesorhizobium gen. nov. Cluster VI harbored a single symbiotic species M. albiziae and is classified as Neomesorhizobium gen. nov. The remaining seven non-symbiotic members were proposed as: Neoaquamicrobium gen. nov., Manganibacter gen. nov., Ollibium gen. nov., Terribium gen. nov., Kumtagia gen. nov., Borborobacter gen. nov., Aerobium gen. nov.. Furthermore, the genus Corticibacterium is restored and two species in Subcluster IX-1 are reclassified as the member of this genus. Conclusion The Mesorhizobium complex are classified into 15 genera based on phylogenomic analyses and OGRIs of 65 type strains. This study resolved previously non-monophyletic genera in the Mesorhizobium complex.

Two Gram-negative, aerobic, rod-shaped bacterial strains, 7MK25T and 6Y81T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Based on the results of 16S rRNA gene sequence analysis, strain 7MK25T showed the highest similarity (93.6 %) to Methyloferula stellata AR4T, followed by Bosea thiooxidans DSM 9653T (93.3 %). Strain 6Y81T had the highest similarity of 97.9 % to Lichenibacterium minor RmlP026T, followed by Lichenibacterium ramalinae RmlP001T (97.2 %). Phylogenomic analysis using the UBCG and PhyloPhlAn methods consistently showed that strain 7MK25T formed a sister clade to Boseaceae, while strain 6Y81T formed an independent clade within the genus Lichenibacterium, both in the order Hyphomicrobiales. The digital DNA–DNA hybridization and average nucleotide identity values between strains 7MK25T, 6Y81T and their close relatives were in the ranges of 19.1–29.9 % and 72.5–85.5 %, respectively. The major fatty acids of 7MK25T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C19 : 0 cyclo ω8c, C16 : 0 and C17 : 0 cyclo, while those of 6Y81T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C16 : 0 and C16 : 0 3-OH. Strains 7MK25T and 6Y81T took diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine as their dominant polar lipids, and Q-10 as their major respiratory quinone. On the basis of phenotypic and phylogenetic data, strain 7MK25T is proposed to represent a novel species of a novel genus with name Terrirubrum flagellatum gen. nov., sp. nov., within a novel family Terrirubraceae fam. nov., with 7MK25T (=KCTC 62738T=GDMCC 1.1452T) as its type strain. Strain 6Y81T represents a novel species in the genus Lichenibacterium, for which the name Lichenibacterium dinghuense sp. nov. (type strain 6Y81T=KACC 21 727T=GDMCC 1.2176T) is proposed. Rhodoblastaceae fam. nov. with Rhodoblastus as the type genus is also proposed to solve the non-monophylectic problem of the family Roseiarcaceae.

Infections with phytoplasma present one of the most significant biotic stresses influencing plant health, growth, and production. The phytoplasma ‘Candidatus Phytoplasma solani’ infects a variety of plant species. This pathogen impacts the physiological and morphological characteristics of plants causing stunting, yellowing, leaf curling, and other symptoms that can lead to significant economic losses. The aim of this study was to determine biochemical changes in peony (Paeonia tenuifolia L.), mint (Mentha × piperita L.), and dill (Anethum graveolens L.) induced by ‘Ca. Phytoplasma solani’ in Serbia as well as to predict the impact of the biotic stress using artificial neural network (ANN) modeling. The phylogenetic position of the Serbian ‘Ca. Phytoplasma solani’ strains originated from the tested hosts using 16S rRNA (peony and carrot strains) and plsC (mint and dill strains) sequences indicated by their genetic homogeneity despite the host of origin. Biochemical parameters significantly differed in asymptomatic and symptomatic plants, except for total anthocyanidins contents in dill and the capacity of peony and mint extracts to neutralize superoxide anions and hydroxyl radicals, respectively. Principal Component Analysis (PCA) showed a correlation between different chemical parameters and revealed a clear separation among the samples. Based on the ANN performance, the optimal number of hidden neurons for the calculation of TS, RG, PAL, LP, NBT, •OH, TP, TT, Tflav, Tpro, Tant, DPPH, and Car was nine (using MLP 8-9-13), as it produced high r2 values (1.000 during the training period) and low SOS values. Developing an effective early warning system for the detection of plant diseases in different plant species is critical for improving crop yield and quality.

Three Gram-stain-negative, aerobic, rod-shaped, non-motile strains (DC2WT, DC25WT, and LKC2W) were isolated from streams in China. Comparisons based on the 16S rRNA gene sequences showed that these three strains share 16S rRNA gene sequence similarity values over 97.0 % with the species of genus Arcicella. There was confusion due to the fact that all species of genera Flectobacillus, Aquirufa, and Sandaracinomonas show 16S rRNA gene sequence similarity of over 90.0 % to the above three strains, but the genus Flectobacillus belongs to the family Spirosomataceae and the genera Aquirufa and Sandaracinomonas belong to the family Cytophagaceae. Observing the phylogenetic trees, strains DC2WT, DC25WT, and LKC2W cluster closely with the species of genus Arcicella, but some species within the families Spirosomataceae and Cytophagaceae are not monophyletic. The phylogenomic tree also showed a confused phylogenetic relationships among these non-monophyletic species. Combining the phylogenetic relationships and average nucleotide identity values, the current taxonomic status of all the genera within the families Spirosomataceae and Cytophagaceae were re-examined. The genera ‘Chryseosolibacter’, ‘Dawidia’, and Chryseotalea should belong to the new family Chryseotaleaceae fam. nov., the genera Arcicella, Flectobacillus, Pseudarcicella, Aquirufa, and Sandaracinomonas should belong to the new family Flectobacillaceae fam. nov., the genera Fluviimonas, Taeseokella, Arcticibacterium, Emticicia, Jiulongibacter, Marinilongibacter, Lacihabitans, and Leadbetterella should belong to the new family Leadbetterellaceae fam. nov., the genus Litoribacter should be reassigned to the family Cyclobacteriaceae, and the genera Arundinibacter and Tellurirhabdus should be reassigned to the family Spirosomataceae. Strains DC2WT and DC25WT are reported to represent two novel species of the genus Arcicella, for which the names Arcicella gelida sp. nov. (type strain DC2WT=GDMCC 1.3209T=KCTC 92559T) and Arcicella lustrica sp. nov. (type strain DC25WT=GDMCC 1.3210T=KCTC 92557T) are proposed.

Abstract Huanglongbing (HLB), associated with the psyllid-vectored phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), is a disease threat to all citrus production worldwide. Currently, there are no sustainable curative or prophylactic treatments available. In this study, we utilized mass spectrometry (MS)-based metabolomics in combination with 3D molecular mapping to visualize complex chemistries within plant tissues to explore how these chemistries change in vivo in HLB-impacted trees. We demonstrate how spatial information from molecular maps of branches and single leaves yields insight into the biology not accessible otherwise. In particular, we found evidence that flavonoid biosynthesis is disrupted in HLB-impacted trees, and an increase in the polyamine, feruloylputrescine, is highly correlated with an increase in disease severity. Based on mechanistic details revealed by these molecular maps, followed by metabolic modeling, we formulated and tested the hypothesis that CLas infection either directly or indirectly converts the precursor compound, ferulic acid, to feruloylputrescine to suppress the antimicrobial effects of ferulic acid and biosynthetically downstream flavonoids. Using in vitro bioassays, we demonstrated that ferulic acid and bioflavonoids are indeed highly bactericidal to CLas, with the activity on par with a reference antibiotic, oxytetracycline, recently approved for HLB management. We propose these compounds should be evaluated as therapeutics alternatives to the antibiotics for HLB treatment. Overall, the utilized 3D metabolic mapping approach provides a promising methodological framework to identify pathogen-specific inhibitory compounds in planta for potential prophylactic or therapeutic applications.