Physiology

Nitrospira -like bacteria are among the most diverse and widespread nitrifiers in natural ecosystems and the dominant nitrite oxidizers in wastewater treatment plants (WWTPs). The recent discovery of comammox-like Nitrospira strains, capable of complete oxidation of ammonia to nitrate, raises new questions about specific traits responsible for the functional versatility and adaptation of this genus to a variety of environments. The availability of new Nitrospira genome sequences from both nitrite-oxidizing and comammox bacteria offers a way to analyze traits in different Nitrospira functional groups. Our comparative genomics analysis provided new insights into the adaptation of Nitrospira strains to specific lifestyles and environmental niches.

Pathogens from the fastidious, phloem-restricted ‘Candidatus Liberibacter’ species cause the devastating Huanglongbing (HLB) disease in citrus worldwide and cause diseases on many solanaceous crops and plants in the Apiaceae family. However, little is known about the pathogenic mechanisms due to the difficulty in culturing the corresponding ‘Ca. Liberibacter’ species. Here, we report that the citrus HLB pathogen ‘Ca. L. asiaticus’ uses an active salicylate hydroxylase SahA to degrade salicylic acid (SA) and suppress plant defenses. Purified SahA protein displays strong enzymatic activity to degrade SA and its derivatives. Overexpression of SahA in transgenic tobacco plants abolishes SA accumulation and hypersensitive response (HR) induced by nonhost pathogen infection. By degrading SA, ‘Ca. L. asiaticus’ not only enhances the susceptibility of citrus plants to both nonpathogenic and pathogenic Xanthomonas citri but also attenuates the responses of citrus plants to exogenous SA. In addition, foliar spraying of 2,1,3-benzothiadiazole and 2,6-dichloroisonicotinic acid, SA functional analogs not degradable by SahA, displays comparable (and even better) effectiveness with SA in suppressing ‘Ca. L. asiaticus’ population growth and HLB disease progression in infected citrus trees under field conditions. This study demonstrates one or more pathogens suppress plant defenses by degrading SA and establish clues for developing novel SA derivatives-based management approaches to control the associated plant diseases.

Huanglongbing, a destructive disease of citrus, is caused by the fastidious bacterium ‘Candidatus Liberibacter asiaticus’ and transmitted by Asian citrus psyllid, Diaphorina citri. The impact of ‘Ca. L. asiaticus’ infection or D. citri infestation on Valencia sweet orange (Citrus sinensis) leaf metabolites was investigated using gas chromatography mass spectrometry, followed by gene expression analysis for 37 genes involved in jasmonic acid (JA), salicylic acid (SA), and proline-glutamine pathways. The total amino acid abundance increased after ‘Ca. L. asiaticus’ infection, while the total fatty acids increased dramatically after infestation with D. citri, compared with control plants. Seven amino acids (glycine, l-isoleucine, l-phenylalanine, l-proline, l-serine, l-threonine, and l-tryptophan) and five organic acids (benzoic acid, citric acid, fumaric acid, SA, and succinic acid) increased in ‘Ca. L. asiaticus’-infected plants. On the other hand, the abundance of trans-JA and its precursor α-linolenic increased in D. citri-infested plants. Surprisingly, the double attack of both D. citri infestation and ‘Ca. L. asiaticus’ infection moderated the metabolic changes in all chemical classes studied. In addition, the gene expression analysis supported these results. Based on these findings, we suggest that, although amino acids such as phenylalanine are involved in citrus defense against ‘Ca. L. asiaticus’ infection through the activation of an SA-mediated pathway, fatty acids, especially α-linolenic acid, are involved in defense against D. citri infestation via the induction of a JA-mediated pathway.

AbstractGrapevine (Vitis vinifera) is one of the most important fruits in Iran where the provinces of Qazvin, Lorestan and Markazi are main producers. During 2013–2015, vineyards located in these provinces were surveyed to verify the presence of phytoplasma. The sample collection was based on symptomatology including decline, leaf yellowing and shortening of internodes. Total DNA was extracted from symptomatic and symptomless grapevine samples and used in nested‐polymerase chain reaction (PCR) assays with phytoplasma ribosomal primers (P1/Tint followed by R16F2n/R2, R16mF1/mR1, R16(I)F1/R1 or 6R758f/16R1232r). Nested‐PCR products were obtained only for symptomatic samples while samples from symptomless plants yielded no PCR products. Restriction fragment length polymorphism (RFLP) analyses with Tru1I, TaqI and Tsp509I and direct sequencing of amplicons followed by phylogenetic analyses indicated the presence of ‘Candidatus Phytoplasma fraxini’, ‘Ca. P. aurantifolia’, ‘Ca. P. solani’ and ‘Ca. P. phoenicium’‐related strains. In Marzaki province, there ‘Ca. P. aurantifolia’ strains were mainly detected, while in the other two provinces, all the four ‘Candidatus species’ were identified with the prevalence of ‘Ca. P. solani’‐related strains. In both provinces in one case, mixed phytoplasma infection was also detected by RFLP analyses. The presence of different phytoplasmas in positive samples indicates great phytosanitary significance due to grapevine economic importance for country. Grapevine phytoplasma infection represents a threat for other crops suggesting grapevine as alternative host species for the phytoplasmas already reported in Iran, while the ‘Ca. P. fraxini’ is for the first time identified in Iran.

Huanglongbing (HLB) is currently the largest threat to global citrus production. We examined the effect of HLB pathogen ‘Candidatus Liberibacter asiaticus’ infection or infestation by its vector, Diaphorina citri, on ‘Valencia’ sweet orange leaf pigments using high-performance liquid chromatography, followed by gene expression analysis for 46 involved genes in carotenoid and chlorophyll biosynthesis pathways. Both ‘Ca. L. asiaticus’ and D. citri alter the total citrus leaf pigment balance with a greater impact by ‘Ca. L. asiaticus’. Although zeaxanthin was accumulated in ‘Ca. L. asiaticus’-infected leaves, chlorophyllide a was increased in D. citri-infested plants. Our findings support the idea that both ‘Ca. L. asiaticus’ and D. citri affect the citrus pigments and promote symptom development but using two different mechanisms. ‘Ca. L. asiaticus’ promotes chlorophyll degradation but accelerates the biosynthesis of carotenoid pigments, resulting in accumulation of abscisic acid and its precursor, zeaxanthin. Zeaxanthin also has a photoprotective role. By contrast, D. citri induced the degradation of most carotenoids and accelerated chlorophyll biosynthesis, leading to chlorophyllide a accumulation. Chlorophyllide a might have an antiherbivory role. Accordingly, we suggest that citrus plants try to defend themselves against ‘Ca. L. asiaticus’ or D. citri using multifaceted defense systems, based on the stressor type. These findings will help in better understanding the tritrophic interactions among plant, pathogen, and vector.

Bacteria that directly use electrodes as metabolic electron donors (biocathodes) have been proposed for applications ranging from microbial electrosynthesis to advanced bioelectronics for cellular communication with machines. However, just as we understand very little about oxidation of analogous natural insoluble electron donors, such as iron oxide, the organisms and extracellular electron transfer (EET) pathways underlying the electrode-cell direct electron transfer processes are almost completely unknown. Biocathodes are a stable biofilm cultivation platform to interrogate both the rate and mechanism of EET using electrochemistry and to study the electroautotrophic organisms that catalyze these reactions. Here we provide new evidence supporting the hypothesis that the uncultured bacterium “CandidatusTenderia electrophaga” directly couples extracellular electron transfer to CO2fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy.

AbstractReddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma‐specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN‐1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII‐A), respectively. The phylogenetic trees showed that LN‐1 phytoplasma clustered with the members of 16SrXII‐A group, including Ca. P. solani. Computer‐simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.

“ Ca . Pelagibacter ubique” is a key driver of marine biogeochemistry cycles and a model for understanding how minimal genomes evolved in free-living anucleate organisms. This study explores the unusual sulfur acquisition strategy that has evolved in these cells, which lack assimilatory sulfate reduction and instead rely on reduced sulfur compounds found in oxic marine environments to meet their cellular quotas. Our findings demonstrate that the sulfur acquisition systems are constitutively expressed but the enzymatic steps leading to the essential sulfur-containing amino acid methionine are regulated by a unique array of riboswitches and genes, many of which are encoded in a rapidly evolving genome region. These findings support mounting evidence that streamlined cells have evolved regulatory mechanisms that minimize transcriptional switching and, unexpectedly, localize essential sulfur acquisition genes in a genome region normally associated with adaption to environmental variation.

Although there are no known sources of genetic resistance, some Citrus spp. are reportedly tolerant to huanglongbing (HLB), presumably caused by ‘Candidatus Liberibacter asiaticus’. Time-course transcriptional analysis of tolerant rough lemon (Citrus jambhiri) and susceptible sweet orange (C. sinensis) in response to ‘Ca. L. asiaticus’ infection showed more genes differentially expressed in HLB-affected rough lemon than sweet orange at early stages but substantially fewer at late time points, possibly a critical factor underlying differences in sensitivity to ‘Ca. L. asiaticus’. Pathway analysis revealed that stress responses were distinctively modulated in rough lemon and sweet orange. Although microscopic changes (e.g., callose deposition in sieve elements and phloem cell collapse) were found in both infected species, remarkably, phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The difference in phloem cell transport activities is also implicated in the differential sensitivity to HLB between the two species. The results potentially lead to identification of key genes and the genetic mechanism in rough lemon to restrain disease development and maintain (or recover) phloem transport activity. These potential candidate genes may be used for improving citrus tolerance (or even resistance) to HLB by genetic engineering.