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"Jiaobai" is a symbiont of Zizania latifolia and Ustilago esculenta, producing fleshy galls as a popular vegetable in South and East Asia. Current "Jiaobai" cultivars exhibit abundant variation in their gall formation date; however, the underlying mechanism is not clear. In this study, a strict short-day (SD) "Jiaobai" line "YD-3" was used. Plants were treated with two day-length regimes [14 h/10 h (day/night) (control) and 8 h/16 h (day/night) (SD)] from 100 to 130 days after planting. The gall swelling rate of the two treatments and another early SD treatment (from 60 to 90 days after planting), together with the contingent flowering plants in the experiment population, revealed that SD can improve both gall enlargement and flowering of "Jiaobai" plants. Comparison of RNA sequencing data among control, SD swelling, and SD flowering treatments of leaves and meristems indicated that SD promotion of "Jiaobai" swelling is conducted by the CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway, similar but not identical to the SD-induced flowering pathway in Z latifolia and rice. "Virus-induced gene silencing", "Yeast one-hybrid assay" and "Dual-luciferase assay" showed that a FT gene, ZlGsd1, is critical in SD promotion of gall formation and is positively regulated by a CO gene, ZlCOL1. Our study elucidated how photoperiod affects the formation of a unique organ produced by plant-fungus symbiosis. The difference in SD response between "Jiaobai" and rice, as well as their potential applications in breeding of "Jiaobai" and rice, were also discussed., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Ustilago maydis is a biotrophic pathogen causing smut disease in maize. It secretes a cocktail of effector proteins, which target different host proteins during its biotrophic stages in the host plant. One such class of proteins we identified previously is TOPLESS (TPL) and TOPLESS-RELATED (TPR) transcriptional corepressors. Here, we screened 297 U. maydis effector candidates for their ability to interact with maize TPL protein RAMOSA 1 ENHANCER LOCUS 2 LIKE 2 (RELK2) and their ability to induce auxin signaling and thereby identified three novel TPL-interacting protein effectors (Tip6, Tip7, and Tip8). Structural modeling and mutational analysis allowed the identification of TPL-interaction motifs of Tip6 and Tip7. In planta interaction between Tip6 and Tip7 with RELK2 occurs mainly in nuclear compartments, whereas Tip8 colocalizes with RELK2 in a compartment outside the nucleus. Overexpression of Tip8 in nonhost plants leads to cell death, indicating recognition of the effector or its activity. By performing infection assays with single and multideletion mutants of U. maydis, we demonstrate a positive role of Tip6 and Tip7 in U. maydis virulence. Transcriptional profiling of maize leaves infected with Tip effector mutants in comparison with SG200 strain suggests Tip effector activities are not merely redundant., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Crown gall caused by Agrobacterium vitis (A. vitis) is one of the crucial issues restricting the to grape industry. In this study, Agrobacterium tumefaciens (Ag-8) was separated from the soil that could prevent the occurrence of grape crown gall. By the mutagenesis of Ag-8 transposon, the siaD gene deletion strain (ΔsiaD) showed significantly lower efficacy in grape and tomato plants for controlling grape crown gall, but the relevant mechanism was not clear. The biofilm formation and motility of ΔsiaD were significantly decreased, and the colonization ability of ΔsiaD in tomato roots was significantly reduced. RNA-seq analysis showed that the expression of nemR significantly reduced in the ΔsiaD and that the expression of nemR showed a high correlation with biofilm and motility. Further studies showed that the nemR gene deletion strain of Ag-8 (ΔnemR) showed significantly reduced motility, biofilm formation and control of grape crown gall compared to Ag-8, and the nemR gene complementary strain of Ag-8 (ΔnemR-comp) recovered to Ag-8 wild-type levels. The inoculation experiments of preventive, curative or simultaneous treatment further showed that the preferential inoculation with Ag-8 reduced the incidence of grape crown gall on tomato plants, and studies showed that the mutation of siaD affected the site competition between Ag-8 and A. vitis, and that the mutation of nemR was consistent with the previous results. This study provides a new strategy for the prevention and control of grape crown gall, which is of great significance to the grape industry to increase production and income., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Despite Corylus avellana L. being an economically important shrub species known for its resilience to adverse environmental conditions, it constantly faces attacks from a plethora of biotic entities. Among these, the mite pest Phytoptus avellanae is gaining importance, causing economic losses every year. This mite colonises the new generative and vegetative buds, leading them to become swollen and reddish, and drastically reducing hazelnut production. The biology behind gall formation is still poorly understood. This study provides a qualitative and quantitative description of the microbiome in both healthy and infested buds of two economically important hazelnut cultivars through metabarcoding of fungal ITS and bacterial 16 S. Potentially pathogenic genera such as Fusarium and Pseudomonas were predominant in the infested buds, along with the obligate intracellular bacterial genus Wolbachia. Akanthomyces muscarius was instead isolated from culture-based methods only from the infested buds. These findings could improve the understanding of gall ecology, supporting the management of mite populations, and they could also serve as a milestone for further studies on low-impact, monitoring-driven, and genetically targeted control strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cerasus × yedoensis (cherry 'Somei-yoshino' Fujino) is affected by bacterial gall disease caused by Pseudomonas syringae pv. cerasicola (PSC). C. × yedoensis is often infected with PSC under weak light intensity, which indicates that susceptibility of C. × yedoensis to PSC is affected by light. To evaluate the effects of white light intensity and different light qualities, white or blue, on bacterial gall disease development, we quantitatively assessed the anatomical and histological features of bacterial-inoculated sites on branches of 2-year-old potted C . × yedoensis seedlings grown under different light intensities and qualities. The stronger the white light intensity, the less severe the gall symptoms. Gall formation was suppressed more by blue than white light of the same intensity. The validity of a simple gall index for assessing gall development with the naked eye, via quantitative evaluation of gall shape by measuring gall height, width, and volume, showed that the gall index could be used as a practical method for on-site assessments of gall development. The ratio of degeneration area in the gall remained constant, suggesting the presence of some regulatory mechanism preventing PSC from affecting the entire gall within the plant. Microscopy showed that the gall tissue is composed primarily of callus cells and has voids containing gummy material that is exuded from cracks in the gall, and the periderm develops at the gall foot but not at the gall apex, so the cells at the gall apex were necrotic or collapsed., Competing Interests: The author(s) declare no conflict of interest.

The pathogen Agrobacterium tumefaciens is known for causing crown gall tumours in plants. However, it has also been harnessed as a valuable tool for plant genetic transformation. Apart from the T-DNA, Agrobacterium also delivers at least five virulence proteins into the host plant cells, which are required for an efficient infection. One of these virulence proteins is VirD5. F-box proteins, encoded in the host plant genome or the Ti plasmid, and the ubiquitin/26S proteasome system (UPS) also play an important role in facilitating Agrobacterium infection. Our study identified two Arabidopsis F-box proteins, D5BF1 and D5BF2, that bind VirD5 and facilitate its degradation via the UPS. Additionally, we found that Agrobacterium partially suppresses the expression of D5BF1 and D5BF2. Lastly, stable transformation and tumorigenesis efficiency assays revealed that D5BF1 and D5BF2 negatively regulate the Agrobacterium infection process, showing that the plant F-box proteins and UPS play a role in defending against Agrobacterium infection., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The capability of entomopathogenic fungi to live as plant endophytes is well established. However, their presence in undiscovered environmental niches represents the beginning of a new challenging research journey. Recently, Akanthomyces muscarius (Ascomycota, Cordycipitaceae) (Petch) Spatafora, Kepler & B. Shrestha was isolated from hazelnut buds infested by the big bud mite pest Phytoptus avellanae Nalepa, which makes the buds swollen, reddish, and unable to further develop. Gall formation is known to be regulated by a consortium of microbes and mites, and to better understand the possible role of A. muscarius within the infested gall, its whole genome sequence was obtained using a hybrid approach of Illumina and Nanopore reads. The functional and comparative genomics analysis provided within this study may help answer questions related to the ecology and the entomopathogenicity of this fungus.

Agrobacterium tumefaciens is a soil-borne pathogenic bacterium that causes crown gall disease in many plants. Chemotaxis offers A. tumefaciens the ability to find its host and establish infection. Being an aerobic bacterium, A. tumefaciens possesses one chemotaxis system with multiple potential chemoreceptors. Chemoreceptors play an important role in perceiving and responding to environmental signals. However, the studies of chemoreceptors in A. tumefaciens remain relatively restricted. Here, we characterized a cytoplasmic chemoreceptor of A. tumefaciens C58 that contains an N-terminal globin domain. The chemoreceptor was designated as Atu1027. The deletion of Atu1027 not only eliminated the aerotactic response of A. tumefaciens to atmospheric air but also resulted in a weakened chemotactic response to multiple carbon sources. Subsequent site-directed mutagenesis and phenotypic analysis showed that the conserved residue His100 in Atu1027 is essential for the globin domain's function in both chemotaxis and aerotaxis. Furthermore, deleting Atu1027 impaired the biofilm formation and pathogenicity of A. tumefaciens . Collectively, our findings demonstrated that Atu1027 functions as an aerotaxis receptor that affects agrobacterial chemotaxis and the invasion of A. tumefaciens into its host., Competing Interests: The authors declare there are no competing interests., (©2024 Huang et al.)

Tailocins are headless phage tail structures that mediate interbacterial antagonism. Although the prototypical tailocins, R- and F-pyocins, in Pseudomonas aeruginosa, and other predominantly R-type tailocins have been studied, their presence in Alphaproteobacteria remains unexplored. Here, we report the first alphaproteobacterial F-type tailocin, named rhizoviticin, as a determinant of the biocontrol activity of Allorhizobium vitis VAR03-1 against crown gall. Rhizoviticin is encoded by a chimeric prophage genome, one providing transcriptional regulators and the other contributing to tail formation and cell lysis, but lacking head formation genes. The rhizoviticin genome retains a nearly intact early phage region containing an integrase remnant and replication-related genes critical for downstream gene transcription, suggesting an ongoing transition of this locus from a prophage to a tailocin-coding region. Rhizoviticin is responsible for the most antagonistic activity in VAR03-1 culture supernatant against pathogenic A. vitis strain, and rhizoviticin deficiency resulted in a significant reduction in the antitumorigenic activity in planta. We identified the rhizoviticin-coding locus in eight additional A. vitis strains from diverse geographical locations, highlighting a unique survival strategy of certain Rhizobiales bacteria in the rhizosphere. These findings advance our understanding of the evolutionary dynamics of tailocins and provide a scientific foundation for employing rhizoviticin-producing strains in plant disease control., (© The Author(s) [2024]. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Pathogenic Agrobacterium tumefaciens and Rhodococcus fascians are phytobacteria that induce crown gall and leafy gall disease, respectively, resulting in undesirable growth abnormalities. When present in nurseries, plants infected by either bacterium are destroyed, resulting in substantial losses for growers, especially those producing plants valued for their ornamental attributes. There are many unanswered questions regarding pathogen transmission on tools used to take cuttings for propagation and whether products used for bacterial disease control are effective. We investigated the ability to transmit pathogenic A. tumefaciens and R. fascians on secateurs and the efficacy of registered control products against both bacteria in vitro and in vivo . Experimental plants used were Rosa × hybrida , Leucanthemum × superbum , and Chrysanthemum × grandiflorum for A. tumefaciens and Petunia × hybrida and Oenothera 'Siskiyou' with R . fascians. In separate experiments, we found secateurs could convey both bacteria in numbers sufficient to initiate disease in a host-dependent manner and that bacteria could be recovered from secateurs after a single cut through an infected stem. In in vivo assays, none of six products tested against A. tumefaciens prevented crown gall disease, although several products appeared promising in in vitro trials. Likewise, four compounds trialed against R . fascians failed to prevent disease. Sanitation and clean planting material remain the primary means of disease management., Competing Interests: The author(s) declare no conflict of interest.

Young grapevines (Vitis vinifera) suffer and eventually can die from the crown gall disease caused by the plant pathogen Allorhizobium vitis (Rhizobiaceae). Virulent members of A. vitis harbor a tumor-inducing plasmid and induce formation of crown galls due to the oncogenes encoded on the transfer DNA. The expression of oncogenes in transformed host cells induces unregulated cell proliferation and metabolic and physiological changes. The crown gall produces opines uncommon to plants, which provide an important nutrient source for A. vitis harboring opine catabolism enzymes. Crown galls host a distinct bacterial community, and the mechanisms establishing a crown gall-specific bacterial community are currently unknown. Thus, we were interested in whether genes homologous to those of the tumor-inducing plasmid coexist in the genomes of the microbial species coexisting in crown galls. We isolated 8 bacterial strains from grapevine crown galls, sequenced their genomes, and tested their virulence and opine utilization ability in bioassays. In addition, the 8 genome sequences were compared with 34 published bacterial genomes, including closely related plant-associated bacteria not from crown galls. Homologous genes for virulence and opine anabolism were only present in the virulent Rhizobiaceae. In contrast, homologs of the opine catabolism genes were present in all strains including the nonvirulent members of the Rhizobiaceae and non-Rhizobiaceae. Gene neighborhood and sequence identity of the opine degradation cluster of virulent and nonvirulent strains together with the results of the opine utilization assay support the important role of opine utilization for cocolonization in crown galls, thereby shaping the crown gall community., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Crown gall disease of grapevines caused by Allorhizobium vitis causes significant damage to vineyards in cold-climate viticulture areas such as Canada and the northern United States. Introduction of the disease into vineyards occurs mainly through planting of infected but asymptomatic nursery material. Because A. vitis is not a regulated pest for import into Canada, no information on the health status of nursery material destined for import into Canada has previously been collected. This study evaluated the health status of ready-to-plant nursery material from domestic and international nurseries in regard to crown gall by determining the abundance of A. vitis in different plant sections via Droplet Digital PCR technology. In addition, different rootstocks from one nursery were compared. Results showed that A. vitis was present in planting material from all nurseries tested. The bacteria were nonuniformly distributed in dormant nursery material, and there was no difference in abundance between the rootstocks tested. In addition, the first A. vitis strain OP-G1 isolated from galls in British Columbia is described. Results showed that a minimum of 5,000 bacterial OP-G1 cells were needed for symptom expression, suggesting that the initiation of symptom development is not based on presence of bacteria in nursery material alone; a minimum threshold is needed, and environmental conditions need to be met., Competing Interests: The author(s) declare no conflict of interest.

Agrobacteria are important plant pathogens responsible for crown/cane gall and hairy root diseases. Crown/cane gall disease is associated with strains carrying tumor-inducing (Ti) plasmids, while hairy root disease is caused by strains harboring root-inducing (Ri) plasmids. In this study, we analyzed the sequences of Ti plasmids of the novel "tumorigenes" clade of the family Rhizobiaceae ("tumorigenes" Ti plasmids), which includes two species, Rhizobium tumorigenes and Rhizobium rhododendri. The sequences of reference Ti/Ri plasmids were also included, which was followed by a comparative analysis of their backbone and accessory regions. The "tumorigenes" Ti plasmids have novel opine signatures compared with other Ti/Ri plasmids characterized so far. The first group exemplified by pTi1078 is associated with production of agrocinopine, nopaline, and ridéopine in plant tumors, while the second group comprising pTi6.2 is responsible for synthesis of leucinopine. Bioinformatic and chemical analyses, including opine utilization assays, indicated that leucinopine associated with pTi6.2 most likely has D,L stereochemistry, unlike the L,L-leucinopine produced in tumors induced by reference strains Chry5 and Bo542. Most of the "tumorigenes" Ti plasmids have conjugative transfer system genes that are unusual for Ti plasmids, composed of avhD4/avhB and traA/mobC/parA regions. Next, our results suggested that "tumorigenes" Ti plasmids have a common origin, but they diverged through large-scale recombination events, through recombination with single or multiple distinct Ti/Ri plasmids. Lastly, we showed that Ti/Ri plasmids could be differentiated based on pairwise Mash or average amino-acid identity distance clustering, and we supply a script to facilitate application of the former approach by other researchers., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Agrobacterium tumefaciens incites the formation of readily visible macroscopic structures known as crown galls on plant tissues that it infects. Records from biologists as early as the 17th century noted these unusual plant growths and began examining the basis for their formation. These studies eventually led to isolation of the infectious agent, A. tumefaciens, and decades of study revealed the remarkable mechanisms by which A. tumefaciens causes crown gall through stable horizontal genetic transfer to plants. This fundamental discovery generated a barrage of applications in the genetic manipulation of plants that is still under way. As a consequence of the intense study of A. tumefaciens and its role in plant disease, this pathogen was developed as a model for the study of critical processes that are shared by many bacteria, including host perception during pathogenesis, DNA transfer and toxin secretion, bacterial cell-cell communication, plasmid biology, and more recently, asymmetric cell biology and composite genome coordination and evolution. As such, studies of A. tumefaciens have had an outsized impact on diverse areas within microbiology and plant biology that extend far beyond its remarkable agricultural applications. In this review, we attempt to highlight the colorful history of A. tumefaciens as a study system, as well as current areas that are actively demonstrating its value and utility as a model microorganism.

The phytopathogenic bacterium Agrobacterium tumefaciens causes crown gall disease in plants, characterized by the formation of tumor-like galls where wounds were present. Nowadays, however, the bacterium and its Ti (tumor-inducing) plasmid is better known as an effective vector for the genetic manipulation of plants and fungi. In this review, I will briefly summarize some of the major discoveries that have led to this bacterium now playing such a prominent role worldwide in plant and fungal research at universities and research institutes and in agricultural biotechnology for the production of genetically modified crops. I will then delve a little deeper into some aspects of Agrobacterium biology and discuss the diversity among agrobacteria and the taxonomic position of these bacteria, the diversity in Ti plasmids, the molecular mechanism used by the bacteria to transform plants, and the discovery of protein translocation from the bacteria to host cells as an essential feature of Agrobacterium -mediated transformation., Competing Interests: The author(s) declare no conflict of interest.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease on a wide range of host species by transferring and integrating a part of its own DNA (T-DNA) into the plant genome. The genes responsible of the above-mentioned processes are well characterized. However, a large number of the mechanisms involved in exploitation and colonization of the galls (also named plant tumors) remain unknown. Due to recent development of "transposon-sequencing" (Tn-Seq) techniques, a high-throughput screening and identification of the different genes involved in such mechanisms is now possible. In this chapter, we describe the detailed methodology used to construct a transposon library in A. tumefaciens and to conduct a Tn-Seq approach to discover genes involved in plant tumor exploitation and colonization., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Main Conclusion: Plant responds to Agrobacterium via three-layered immunity that determines its susceptibility or resistance to Agrobacterium infection. Agrobacterium tumefaciens is a soil-borne Gram-negative bacterium that causes crown gall disease in plants. The remarkable feat of interkingdom gene transfer has been extensively utilised in plant biotechnology to transform plant as well as non-host systems. In the past two decades, the molecular mode of the pathogenesis of A. tumefaciens has been extensively studied. Agrobacterium has also been utilised as a premier model to understand the defence response of plants during plant-Agrobacterium interaction. Nonetheless, the threat of Agrobacterium-mediated crown gall disease persists and is associated with a huge loss of plant vigour in agriculture. Understanding the molecular dialogues between these two interkingdom species might provide a cure for crown gall disease. Plants respond to A. tumefaciens by mounting a three-layered immune response, which is manipulated by Agrobacterium via its virulence effector proteins. Comparative studies on plant defence proteins versus the counter-defence of Agrobacterium have shed light on plant susceptibility and tolerance. It is possible to manipulate a plant's immune system to overcome the crown gall disease and increase its competence via A. tumefaciens-mediated transformation. This review summarises the recent advances in the molecular mode of Agrobacterium pathogenesis as well as the three-layered immune response of plants against Agrobacterium infection., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The volatile organic compounds (VOCs) produced by endophytic bacteria have a significant role in the control of phytopathogens. In this research, the VOCs produced by endophytic bacteria including Serratia sp. Ba10, Pantoea sp. Sa14, Enterobacter sp. Ou80, Pseudomonas sp. Ou22, Pseudomonas sp. Sn48 and Pseudomonas sp. Ba35, which were previously isolated from healthy domesticated and wild-growing grapevine were evaluated in terms of their effects on the virulence traits of Agrobacterium tumefaciens Gh1, the causal agent of crown gall disease. Based on the gas chromatography-mass spectrometry analysis, 16, 15, 14, 7, 16, and 15 VOCs have been identified with high quality in strains of Ba10, Sa14, Ou80, Ou22, Sn48, and Ba35, respectively. All endophytic bacteria produced VOCs that significantly reduced crown gall symptoms and inhibited the populations of A. tumefaciens Gh1 at different levels. Moreover, scanning electron microscopy analysis revealed various morphological abnormalities in the A. tumefaciens cells exposed to the VOCs produced by Ba35, Ou80, and Sn48 strains. The VOCs significantly reduced swarming-, swimming-, twitching motility and biofilm formation by A. tumefaciens Gh1. Our results revealed that VOCs could reduce the attachment of A. tumefaciens Gh1 cells to root tissues of grapevine cultivars Rashe and Bidane sefid, as well as chemotaxis motility towards root extract of both cultivars. Based on our results, it was shown that the antibacterial VOCs produced by endophytic bacteria investigated in the current study can manage crown gall disease and increase our knowledge on the role of VOCs in microbial interactions., (© 2022. The Author(s).)

Many plant-associated fungi are obligate biotrophs that depend on living hosts to proliferate. However, little is known about the molecular basis of the biotrophic lifestyle, despite the impact of fungi on the environment and food security. In this work, we show that combinations of organic acids and glucose trigger phenotypes that are associated with the late stage of biotrophy for the maize pathogen Ustilago maydis . These phenotypes include the expression of a set of effectors normally observed only during biotrophic development, as well as the formation of melanin associated with sporulation in plant tumors. U. maydis and other hemibiotrophic fungi also respond to a combination of carbon sources with enhanced proliferation. Thus, the response to combinations of nutrients from the host may be a conserved feature of fungal biotrophy.

The genus Xanthomonas contains a set of diverse bacterial strains, most of which are known for their pathogenicity on annual crops and fruit trees causing economically important plant diseases. Recently, five Xanthomonas strains were isolated from Agrobacterium -induced crown gall tissues of amaranth ( Amaranthus sp.) and weeping fig ( Ficus benjamina ) plants in Iran. Phenotypic characteristics ( i.e . biochemical tests and pathogenicity features) and whole genome sequence-based core-genome phylogeny followed by average nucleotide identity and digital DNA-DNA hybridization calculations suggested that these gall-associated strains belong to two new species within the genus Xanthomonas . In this study, we provide a formal species description for these new species where Xanthomonas bonasiae sp. nov. is proposed for the strains isolated from weeping fig with FX4 T (=CFBP 8703 T =DSM 112530 T ) as type strain. The name Xanthomonas youngii sp. nov. is proposed for the strains isolated from amaranth with AmX2 T (=CFBP 8902 T =DSM 112529 T ) as type strain.

Agrobacterium tumefaciens species complex contains a set of diverse bacterial strains, most of which are well known for their pathogenicity on agricultural plants causing crown gall diseases. Members of A. tumefaciens species complex are classified into several taxonomically distinct lineages called "genomospecies" (13 genomospecies until early 2021). Recently, two genomospecies, G19 (strains Rnr T , Rew, and Rnw) and G20 (strains OT33 T and R13) infecting Rosa sp. plants in Iran, were described based on biochemical and molecular-phylogenetic data. Whole genome sequence-based core-genome phylogeny followed by average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) calculations performed in this study suggested that genomospecies G19 and G20 could be described as two novel and standalone species. In the phylogenetic tree, these two new genomospecies were clustered separately from other genomospecies/species of A. tumefaciens species complex. Moreover, both ANI and dDDH indices between the G19/G20 strains and other Rhizobiaceae members are clearly below the accepted thresholds for prokaryotic species description. Hence, Agrobacterium burrii sp. nov. is proposed to encompass the G19 strains, with Rnr T = CFBP 8705 T = DSM 112541 T as type strain. Agrobacterium shirazense sp. nov. is also proposed to include G20 strains, with OT33 T = CFBP 8901 T = DSM 112540 T as type strain.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

In summer 2019, widespread occurrence of crown gall disease caused by Agrobacterium spp. was observed on commercially grown ornamental plants in southern Iran. Beside agrobacteria, pale yellow-pigmented Gram-negative strains resembling the members of Xanthomonas were also associated with crown gall tissues on weeping fig (Ficus benjamina) and Amaranthus sp. plants. The purpose of the present study was to characterize the crown gall-associated Xanthomonas strains using plant inoculation assays, molecular-phylogenetic analyses, and comparative genomics approaches. Pathogenicity tests showed that the Xanthomonas strains did not induce disease symptoms on their host of isolation. However, the strains induced hypersensitive reaction on tobacco, geranium, melon, squash, and tomato leaves via leaf infiltration. Multilocus sequence analysis suggested that the strains belong to clade IA of Xanthomonas , phylogenetically close to Xanthomonas translucens, X. theicola, and X. hyacinthi. Average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of the strains isolated in this study and reference Xanthomonas strains are far below the accepted thresholds for the definition of prokaryotic species, signifying that these strains could be defined as two new species within clade IA of Xanthomonas . Comparative genomics showed that the strains isolated from crown gall tissues are genetically distinct from X. translucens , as almost all the type III secretion system genes and type III effectors are lacking in the former group. The data obtained in this study provide novel insight into the breadth of genetic diversity of crown gall-associated bacteria and pave the way for research on gall-associated Xanthomonas -plant interactions. IMPORTANCE Tumorigenic agrobacteria-members of the bacterial family Rhizobiaceae -cause crown gall and hairy root diseases on a broad range of plant species. These bacteria are responsible for economic losses in nurseries of important fruit trees and ornamental plants. The microclimate of crown gall and their accompanying microorganisms has rarely been studied for the microbial diversity and population dynamics of gall-associated bacteria. Here, we employed a series of biochemical tests, pathogenicity assays, and molecular-phylogenetic analyses, supplemented with comparative genomics, to elucidate the biological features, taxonomic position, and genomic repertories of five crown gall-associated Xanthomonas strains isolated from weeping fig and Amaranthus sp. plants in Iran. The strains investigated in this study induced hypersensitive reactions (HR) on geranium, melon, squash, tobacco, and tomato leaves, while they were nonpathogenic on their host of isolation. Phylogenetic analyses and whole-genome-sequence-based average nucleotide identity (ANI)/digital DNA-DNA hybridization (dDDH) calculations suggested that the Xanthomonas strains isolated from crown gall tissues belong to two taxonomically unique clades closely related to the clade IA species of the genus, i.e., X. translucens , X. hyacinthi , and X. theicola .

Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants. Transposon-sequencing (Tn-Seq) was used to discover A.tumefaciens genes involved in reproductive success (fitness genes) on Solanum lycopersicum and Populus trichocarpa tumors and S.lycopersicum and Zea mays roots. The identified fitness genes represent 3-8% of A. tumefaciens genes and contribute to carbon and nitrogen metabolism, synthesis and repair of DNA, RNA and proteins and envelope-associated functions. Competition assays between 12 knockout mutants and wild-type confirmed the involvement of 10 genes (trpB, hisH, metH, cobN, ntrB, trxA, nrdJ, kamA, exoQ, wbbL) in A.tumefaciens fitness under both tumor and root conditions. The remaining two genes (fecA, noxA) were important in tumors only. None of these mutants was nonpathogenic, but four (hisH, trpB, exoQ, ntrB) exhibited impaired virulence. Finally, we used this knowledge to search for chemical and biocontrol treatments that target some of the identified fitness pathways and report reduced tumorigenesis and impaired establishment of A.tumefaciens on tomato roots using tannic acid or Pseudomonas protegens, which affect iron assimilation. This work revealed A.tumefaciens pathways that contribute to its competitive survival in plants and highlights a strategy to identify plant protection approaches against this pathogen., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Kiwifruit is moderately sweet and sour and quite popular among consumers; it has been widely planted in some areas of the world. In 2019, the crown gall disease of kiwifruit was discovered in the main kiwifruit-producing area of Guizhou Province, China. This disease can weaken and eventually cause the death of the tree. The phylogeny, morphological and biological characteristics of the bacteria were described, and were related to diseases. The pathogenicity of this species follows the Koch hypothesis, confirming that A. fabacearum is the pathogen of crown gall disease of kiwifruit in China. In this study, Loop-mediated isothermal amplification (LAMP) analysis for genome-specific gene sequences was developed for the specific detection of A. fabacearum . The detection limit of the LAMP method is 5 × 10 -7 ng/μL, which has high sensitivity. At the same time, the amplified product is stained with SYBR Green I after the reaction is completed, so that the amplification can be detected with the naked eye. LAMP analysis detected the presence of A. fabacearum in the roots and soil samples of the infected kiwifruit plant. The proposed LAMP detection technology in this study offers the advantages of ease of operation, visibility of results, rapidity, accuracy and high sensitivity, making it suitable for the early diagnosis of crown gall disease of kiwifruit.

The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta , which may conduct fungus growth and infection at the initial stage of stem-gall formation.

Background: The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors., Results: We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin., Conclusions: The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium., (© 2021. The Author(s).)

Ustilago maydis is a biotrophic fungus causing smut disease in corn. The infectious forms are dikaryotic hyphae. Here we analyze mutants lacking the nlt1 transcription factor and investigate why these mutants are unable to induce leaf tumors. The study involved reverse genetics, complementation, epistasis analysis, microscopy, gene expression analysis by quantitative reverse transcriptase PCR and virulence assays. We show that nlt1 mutants colonize maize leaves efficiently but fail to undergo karyogamy and are attenuated in late proliferation. Nlt1 activates transcription of ros1, a transcription factor controlling karyogamy, and represses see1, an effector previously shown to contribute to leaf tumor induction. In mononuclate solopathogenic strains, nlt1 mutants cause attenuated leaf tumor formation. In actively dividing maize organs, nlt1 mutants undergo karyogamy and induce tumor formation. Sporisorium reilianum, a smut fungus unable to induce leaf tumors, possesses an ortholog of nlt1 that controls the fusion of dikaryotic nuclei late in infection during cob colonization. Our results have established a regulatory connection between nlt1, ros1 and see1 and suggest the existence of two stages contributing to leaf tumor formation, one before nuclear fusion and involving nlt1 and one after karyogamy that is nlt1 independent., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

The reduction of the use chemical pesticides in agriculture is gaining importance as an objective of decision-makers in both politics and economics. Consequently, the development of technically efficient and economically affordable alternatives as, e.g., biological control agents or practices is highly solicited. Crown gall disease of dicotyledonous plants is caused by ubiquitous soil borne pathogenic bacteria of the Agrobacterium tumefaciens species complex, that comprises the species Agrobacterium fabrum and represents a globally relevant plant protection problem. Within the framework of a screening program for bacterial Agrobacterium antagonists a total of 14 strains were isolated from Tunisian soil samples and assayed for antagonistic activity against pathogenic agrobacteria. One particularly promising isolate, termed strain MBY2, was studied more in depth. Using a Multilocus Sequence Analysis (MLSA) approach, the isolate was assigned to the taxonomic species Bacillus velezensis. Strain MBY2 was shown to display antagonistic effects against the pathogenic A. fabrum strain C58 in vitro and to significantly decrease pathogen populations under sterile and non-sterile soil conditions as well as in the rhizosphere of maize and, to a lower extent, tomato plants. Moreover, the ability of B. velezensis MBY2 to reduce C58-induced gall development has been demonstrated in vivo on stems of tomato and almond plants. The present study describes B. velezensis MBY2 as a newly discovered strain holding potential as a biological agent for crown gall disease management., Competing Interests: The authors have declared that no competing interests exist.

Beneficial rhizobacteria can inhibit soilborne pathogens by secreting an array of polyketides, lipopeptides and dipeptides, but the effect of polyketides on crown gall disease caused by Agrobacterium tumefaciens C58 is unclear. In this study, the antagonistic compounds of the plant growth-promoting rhizobacterium Bacillus velezensis CLA178 was sorted with different organic phases, purified by high-pressure liquid chromatography, and detected by a liquid chromatography ionization-mass spectrometry system. Macrolactins were found to be the compounds with antagonistic activity against A. tumefaciens C58. When the macrolactin synthesis pathway was disrupted, the mutant △mlnA only showed slight antagonistic activity against A. tumefaciens C58. Transmission electron microscopy showed that the inhibition of C58 cell division by cell-free culture from the mutant △mlnA was weaker than that by cell-free culture from CLA178. The mutant deficient in production of macrolactin showed a weaker transcription of genes involved in attachment of C58 to plant and lower biocontrol of crown gall disease in rose than the wild-type strain CLA178. The effect of macrolactins on pathogen C58 has been also confirmed by the purified macrolactins. These results reveal that macrolactins contribute to the biocontrol activity of C58 by inhibiting cell division and downregulating the transcription of chvB and chvE.

The plant pathogen Agrobacterium tumefaciens causes crown gall disease and is a widely used tool for generating transgenic plants owing to its virulence. The pathogenic process involves a shift from an independent to a living form within a host plant. However, comprehensive analyses of metabolites, genes, and reactions contributing to this complex process are lacking. To gain new insights about the pathogenicity from the viewpoints of physiology and cellular metabolism, a genome-scale metabolic model (GSMM) was reconstructed for A. tumefaciens. The model, referred to as iNX1344, contained 1,344 genes, 1,441 reactions, and 1,106 metabolites. It was validated by analyses of in silico cell growth on 39 unique carbon or nitrogen sources and the flux distribution of carbon metabolism. A. tumefaciens metabolic characteristics under three ecological niches were modelled. A high capacity to access and metabolize nutrients is more important for rhizosphere colonization than in the soil, and substantial metabolic changes were detected during the shift from the rhizosphere to tumour environments. Furthermore, by integrating transcriptome data for tumour conditions, significant alterations in central metabolic pathways and secondary metabolite metabolism were identified. Overall, the GSMM and constraint-based analysis could decode the physiological and metabolic features of A. tumefaciens as well as interspecific interactions with hosts, thereby improving our understanding of host adaptation and infection mechanisms., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Main Conclusion: The presence of Bacillus cereus plays a key role in clubroot suppression and improves plant biomass in pak choi. B. cereus is reported for the first time as a novel biocontrol agent against clubroot. Plasmodiophora brassicae Woronin causes a devastating infectious disease known as clubroot that is damaging to cruciferous vegetables. This study aimed to isolate beneficial bacteria from the rhizosphere soil of pak choi (Brassica campestris sp. chinensis) and to evaluate the ability of the isolate to reduce the severity of clubroot. Strains obtained from the rhizosphere of symptomless pak choi were first selected on the basis of their germination inhibition rate and effects on the viability of P. brassicae resting spores. Eight bacterial isolates had inhibitory effects against the resting spores of clubroot causing pathogen. However, MZ-12 showed the highest inhibitory effect at 73.4%. Inoculation with MZ-12 enhanced the plant biomass relative to plants grown without MZ-12 as well as P. brassicae infected plants. Furthermore, enhanced antioxidant enzymatic activities were observed in clubroot-infected plants during bacterial association. Co-inoculation of the plant with both P. brassicae and MZ-12 resulted in a 64% reduction of gall formation in comparison to plants inoculated with P. brassicae only. Three applications of MZ-12 to plants infected with P. brassicae at 7, 14 and 21 days after seeding (DAS) were more effective than one application and repressed root hair infection. According to 16S rDNA sequence analysis, strain MZ-12 was identified as had a 100% sequence similarity with type strain Bacillus cereus. The findings of the present study will facilitate further investigation into biological mechanisms of cruciferous clubroot control.

Reliable methods of phenotype prediction from genomic data play an increasingly important role in many areas of plant and animal breeding. Thus, developing methods that enhance prediction accuracy is of major interest. Here, we provide three methods for this purpose: (1) Genomic Best Linear Unbiased Prediction (GBLUP) as a model just accounting for additive SNP effects; (2) Epistatic Random Regression BLUP (ERRBLUP) as a full epistatic model which incorporates all pairwise SNP interactions, and (3) selective Epistatic Random Regression BLUP (sERRBLUP) as an epistatic model which incorporates a subset of pairwise SNP interactions selected based on their absolute effect sizes or the effect variances, which is computed based on solutions from the ERRBLUP model. We compared the predictive ability obtained from GBLUP, ERRBLUP, and sERRBLUP with genotypes from a publicly available wheat dataset and respective simulated phenotypes. Results showed that sERRBLUP provides a substantial increase in prediction accuracy compared to the other methods when the optimal proportion of SNP interactions is kept in the model, especially when an optimal proportion of SNP interactions is selected based on the SNP interaction effect sizes. All methods described here are implemented in the R-package EpiGP, which is able to process large-scale genomic data in a computationally efficient way.

Phlebopus roseus is described as new based on collections from southwest China. Phylogenetic analyses of nuclear rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and portions of nuclear 28S rDNA (28S), translation elongation factor 1-alpha ( tef1 ), and the largest and second largest subunits of RNA polymerase II ( rpb1, rpb2 ) support P. roseus as a novel species in the genus Phlebopus (Boletinellaceae, Boletales). The new species resembles P. portentosus but differs from it in that mature basidiomata have a bright rose-red-colored stipe and a radiate tubular hymenophore with nested pores. Despite extensive searching, P. roseus has only been found at four sites within a 24-hectare orchard dominated by Eriobotrya japonica , which is agriculturally important given its fruit production (loquats). Therefore, this species appears to be endemic and geographically restricted. The ecology of this bolete is also unique. In line with the trophic behavior of other species in the Boletinellaceae, our observations indicate that P. roseus forms a symbiotic association with the scale insect Coccus hesperidum , identified through sequence analysis of its mitochondrial cytochrome c oxidase subunit I ( COI ) region, to form fungus-insect galls that develop on roots of E. japonica trees. Phlebopus roseus is an edible mushroom species and is collected from the type location by farmers and sold commercially in limited quantities at local markets alongside P. portentosus and other fungi.

Rhodococcus Fascians: is a gram-positive phytopathogen that infects a wide range of plant species. The actinomycete induces the formation of neoplastic growths, termed leafy galls, that consist of a gall body covered by small shoots of which the outgrowth is arrested due to an extreme form of apical dominance. In our previous work, we demonstrated that in the developing gall, auxin drives the transdifferentiation of parenchyma cells into vascular elements. In this work, with the use of transgenic Arabidopsis thaliana plants carrying molecular reporters for cell division ( pCYCB1;1:GUS ) and meristematic activity ( pSTM:GUS ), we analyzed the fate of cells within the leafy gall. Our results indicate that the size of the gall body is determined by ongoing mitotic cell divisions as illustrated by strong CYCB1;1 expression combined with the de novo formation of new meristematic areas triggered by STM expression. The shoot meristems that develop in the peripheral parts of the gall are originating from high ectopic STM expression. Altogether the presented data provide further insight into the cellular events that accompany the development of leafy galls in response to R. fascians infection.

Rhizobium vitis strain VAT03-9 (MAFF 211676) is a causal agent of crown gall disease in grapevine. It is one of the pathogenic strains of R. vitis isolated from graft unions of grapevine in Okayama Prefecture, Japan. Inoculation tests verified its virulence for gall formation on grapevine, tomato, and sunflower. It harbors tumor-inducing plasmid. Here, we present the complete genome sequence with annotation of R. vitis VAR03-9 obtained by assembling reads from PacBio and Illumina-sequencers. This genome sequence should be useful for the analyses of pathogenicity and evolutionary lineage of the pathogens of crown gall disease.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rhizobium ( Agrobacterium ) is one genus in the family Rhizobiaceae. Most of the species are epi- or endophytic bacteria which include tumorigenic or rhizogenic pathogens, root nodule bacteria, and commensal endosymbionts. Rhizobium vitis strain VAR06-30 is a commensal bacterium without pathogenicity which was isolated from a rootstock of grapevine in Japan. It also does not have antagonistic activity to the pathogenic strain of R. vitis . Here, we show the complete genome sequence data with annotation of R. vitis VAR06-30 which was analyzed by sequence reads obtained from both PacBio and Illumina platforms. This genome sequence would contribute to the understanding of evolutionary lineage and characteristics of Rhizobium commensal bacteria.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ye et al.)

Background: Phlebopus portentosus and mealy bugs form a fungus-insect gall on the roots of host plants. The fungus and mealy bugs benefit mutually through the gall, which is the key link in the nutritional mechanism of P. portentosus. The cavity of the fungus-insect gall provides an ideal shelter for mealy bugs survival and reproduction, but how does P. portentosus benefit from this symbiotic relationship?, Methodology and Results: Anatomical examination of fungus-insect galls revealed that one or more mealy bugs of different generations were living inside the galls. The mealy bug's mouthpart could penetrate through the mycelium layer of the inside of the gall and suck plant juice from the host plant root. Mealy bugs excreted honeydew inside or outside the galls. The results of both honeydew agar medium and quartz tests showed that the honeydew can attract and promote the mycelial growth of P. portentosus. A test of the relationship between the honeydew and the formation of the fungus-insect gall showed that honeydew promoted gall formation., Conclusions: All experimental results in this study show that the honeydew secreted by mealy bugs can attract and promote the mycelial growth of P. portentosus, forming a fungus-insect gall, because mealy bugs' honeydew is rich in amino acids and sugars., Competing Interests: The authors have declared that no competing interests exist.

Ustilago esculenta, a smut fungus, can induce the formation of culm galls in Zizania latifolia, a vegetable consumed in many Asian countries. Specifically, the mycelia-teliospore (M-T) strain of U. esculenta induces the Jiaobai (JB) type of gall, while the teliospore (T) strain induces the Huijiao (HJ) type. The underlying molecular mechanism responsible for the formation of the two distinct types of gall remains unclear. Our results showed that most differentially expressed genes relevant to effector proteins were up-regulated in the T strain compared to those in the M-T strain during gall formation, and the expression of teliospore formation-related genes was higher in the T strain than the M-T strain. Melanin biosynthesis was also clearly induced in the T strain. The T strain exhibited stronger pathogenicity and greater teliospore production than the M-T strain. We evaluated the implications of the gene regulatory networks in the development of these two type of culm gall in Z. latifolia infected with U. esculenta and suggested potential targets for genetic manipulation to modify the gall type for this crop., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Expression of the tumor-inducing (Ti) plasmid virulence genes of Agrobacterium tumefaciens is required for the transfer of DNA from the bacterium into plant cells, ultimately resulting in the initiation of plant tumors. The vir genes are induced as a result of exposure to certain phenol derivatives, monosaccharides, and low pH in the extracellular milieu. The soil, as well as wound sites on a plant-the usual site of the virulence activity of this bacterium-can contain these signals, but vir gene expression in the soil would be a wasteful utilization of energy. This suggests that mechanisms may exist to ensure that vir gene expression occurs only at the higher concentrations of inducers typically found at a plant wound site. In a search for transposon-mediated mutations that affect sensitivity for the virulence gene-inducing activity of the phenol, 3,5-dimethoxy-4-hydroxyacetophenone (acetosyringone [AS]), an RND-type efflux pump homologous to the MexE/MexF/OprN pump of Pseudomonas aeruginosa was identified. Phenotypes of mutants carrying an insertion or deletion of pump components included hypersensitivity to the vir -inducing effects of AS, hypervirulence in the tobacco leaf explant virulence assay, and hypersensitivity to the toxic effects of chloramphenicol. Furthermore, the methoxy substituents on the phenol ring of AS appear to be critical for recognition as a pump substrate. These results support the hypothesis that the regulation of virulence gene expression is integrated with cellular activities that elevate the level of plant-derived inducers required for induction so that this occurs preferentially, if not exclusively, in a plant environment. IMPORTANCE Expression of genes controlling the virulence activities of a bacterial pathogen is expected to occur preferentially at host sites vulnerable to that pathogen. Host-derived molecules that induce such activities in the plant pathogen Agrobacterium tumefaciens are found in the soil, as well as in the plant. Here, we tested the hypothesis that mechanisms exist to suppress the sensitivity of Agrobacterium species to a virulence gene-inducing molecule by selecting for mutant bacteria that are hypersensitive to its inducing activity. The mutant genes identified encode an efflux pump whose proposed activity increases the concentration of the inducer necessary for vir gene expression; this pump is also involved in antibiotic resistance, demonstrating a relationship between cellular defense activities and the control of virulence in Agrobacterium ., (Copyright © 2020 American Society for Microbiology.)

Main Conclusion: The review provides information on the mechanisms underlying the development of spontaneous and pathogen-induced tumors in higher plants. The activation of meristem-specific regulators in plant tumors of various origins suggests the meristem-like nature of abnormal plant hyperplasia. Plant tumor formation has more than a century of research history. The study of this phenomenon has led to a number of important discoveries, including the development of the Agrobacterium-mediated transformation technique and the discovery of horizontal gene transfer from bacteria to plants. There are two main groups of plant tumors: pathogen-induced tumors (e.g., tumors induced by bacteria, viruses, fungi, insects, etc.), and spontaneous ones, which are formed in the absence of any pathogen in plants with certain genotypes (e.g., interspecific hybrids, inbred lines, and mutants). The causes of the transition of plant cells to tumor growth are different from those in animals, and they include the disturbance of phytohormonal balance and the acquisition of meristematic characteristics by differentiated cells. The aim of this review is to discuss the mechanisms underlying the development of most known examples of plant tumors.

Aims: Crown gall, a phytobacteriosis characterized by the formation of tumours on plant roots was observed in recently planted vineyards of the Meknes region (Morocco). The objective of this research was to analyse the diversity of pathogenic agrobacteria isolated from grapevine in Morocco., Methods and Results: Eighty-two isolates from 11 affected vineyards were characterized by recA sequencing and were found to belong to Agrobacterium tumefaciens genomospecies G1, G4 or G7, Rhizobium rhizogenes, and to Allorhizobium vitis. Only the All. vitis isolates appeared to be pathogenic on tomato and multilocus sequence analysis phylogenetic analyses revealed a weak genetic diversity, with the definition of only four genomic groups. Definition of the All. vitis genomic groups correlated with specific pathogenic traits: indeed, genomic groups differed with respect to the severity of hypersensitive response symptoms on tobacco leaves, the intensity of necrotic response on grapevine explants and opine profiles. Both vitopine and octopine were detected by UHPLC in tumours induced by isolates of three genomic groups, an opine signature scarcely ever reported., Conclusions: Allorhizobium vitis is the only causative agent of crown gall on grape in Morocco, pathogenic isolates can be separated into four genomic groups., Significance and Impact of the Study: This study of recently crown-gall-infested vineyards demonstrated that All. vitis is the only causative agent and revealed the presence of nonpathogenic Agrobacterium strain within tumours. Moreover, as the genetic diversity of the All. vitis isolates is relatively narrow, this study lays the basis for further analyses on the evolution of the disease, on the dissemination of the pTi and more globally on the fate of the different genomic groups in this newly colonized environment., (© 2019 The Society for Applied Microbiology.)

Agrobacterium tumefaciens pathogens use specific compounds denoted opines as nutrients in their plant tumor niche. These opines are produced by the host plant cells genetically modified by agrobacteria. They are imported into bacteria via solute-binding proteins (SBPs) in association with ATP-binding cassette transporters. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. Structural and affinity data on mannopinic acid bound to SBPs are currently lacking while those of the three others mannityl opines are available. We investigated the molecular basis of two pathways for mannopinic acid uptake. MoaA was proposed as the specific SBP for mannopinic acid import in mannityl opines-assimilating agrobacteria, which was validated here using genetic studies and affinity measurements. We structurally characterized the mannopinic acid-binding mode of MoaA in two crystal forms at 2.05 and 1.57 Å resolution. We demonstrated that the non-specific SBP MotA, so far characterized as mannopine and Amadori compound importer, was also able to transport mannopinic acid. The structure of MotA bound to mannopinic acid at 2.2 Å resolution defines a different mannopinic acid-binding signature, similar to that of mannopine. Combining in vitro and in vivo approaches, this work allowed us to complete the characterization of the mannityl-opines assimilation pathways, highlighting the important role of two dual imports of agropinic and mannopinic acids. Our data shed new light on how the mannityl-opines contribute to the establishment of the ecological niche of agrobacteria from the early to the late stages of tumor development., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Clubroot, caused by Plasmodiophora brassicae , is an important disease on canola in Alberta, Canada. The pathogen is grouped into pathotypes according to their virulence reaction on differential hosts. Multiple pathotypes or strains are known exist in one field, one plant, or even one gall. This study was conducted with the objective of testing the prevalence of the coexistence of multiple strains in a single gall. In all, 79 canola clubroot galls were collected from 22 fields across Northern Alberta in 2018. Genomic DNA extracted from these single galls was analyzed using RNase H-dependent PCR (rhPCR). The rhPCR primers were designed to amplify a partial sequence of a dimorphic gene, with one primer pair specific to one sequence and the other primer pair specific to the alternative sequence. The amplification of both sequences from DNA obtained from a single gall would indicate that it contains two different P. brassicae strains. The rhPCR analyses indicated that the P. brassicae populations in 50 of the 79 galls consisted of more than one strain. This result emphasizes the need for cautious interpretation of results when a single-gall population is subject to pathotyping or being used as inoculum in plant pathology research. It also confirms that the maintenance of pathotype diversity within single root galls is a common occurrence which has implications for the durability, and stewardship, of single-gene host resistance.

Bacterial flagella are perceived by the innate immune systems of plants 1 and animals 2 alike, triggering resistance. Common to higher plants is the immunoreceptor FLAGELLIN-SENSING 2 (FLS2) 3 , which detects flagellin via its most conserved epitope, flg22. Agrobacterium tumefaciens, which causes crown gall disease in many crop plants, has a highly diverged flg22 epitope and evades immunodetection by plants so far studied. We asked whether, as a next step in this game of 'hide and seek', there are plant species that have evolved immunoreceptors with specificity for the camouflaged flg22 Atum of A. tumefaciens. In the wild grape species Vitis riparia, we discovered FLS2 XL , a previously unknown form of FLS2, that provides exquisite sensitivity to typical flg22 and to flg22 Atum . As exemplified by ectopic expression in tobacco, FLS2 XL can limit crown gall disease caused by A. tumefaciens.

Agrobacterium-mediated plant galls are often misdiagnosed as nematode-mediated knots, even by experts, because the gall symptoms in both conditions are very similar. In the present study, we developed biosensor strains based on agrobacterial opine metabolism that easily and simply diagnoses Agrobacterium-induced root galls. Our biosensor consists of Agrobacterium mannitol (ABM) agar medium, X-gal, and a biosensor. The working principle of the biosensor is that exogenous nopaline produced by plant root galls binds to NocR, resulting in NocR/nopaline complexes that bind to the promoter of the nopaline oxidase gene (nox) operon and activate the transcription of noxB-lacZY, resulting in readily visualized blue pigmentation on ABM agar medium supplemented with X-gal (ABMX-gal). Similarly, exogenous octopine binds to OccR, resulting in OoxR/octopine complexes that bind to the promoter of the octopine oxidase gene (oox) operon and activate the transcription of ooxB-lacZY, resulting in blue pigmentation in the presence of X-gal. Our biosensor is successfully senses opines produced by Agrobacterium-infected plant galls, and can be applied to easily distinguish Agrobacterium crown gall disease from nematode disease.

Nest architecture is a fundamental character shaping immune strategies of social insects. The arboreal ant Temnothorax unifasciatus nests in cavities such as oak galls where the entire colony lives in a unique small chamber. In these conditions, physiological and behavioural strategies likely prevail over compartmentalisation and are presumably tuned with colony size. We designed two experiments to study chemical and behavioural immune strategies against the entomopathogenic fungus Metarhizium anisopliae in colonies of different sizes. First, we compared spore germination and length of germinal tubes inside artificial nests, designed to impede the contact between the ants and the fungus, in colonies of different size. In the absence of direct contact, Temnothorax unifasciatus colonies inhibit fungal growth inside their nests, presumably through volatile compounds. The analysis revealed a positive correlation between fungistatic activity and colony size, indicating that workers of smaller colonies do not invest a higher per capita effort in producing such substances compared to larger colonies. Second, we performed a removal experiment of contaminated and non-contaminated items introduced inside the nests of colonies of different size. Small colonies challenged with contaminated fibres showed an increased removal of all the items (both contaminated and non-contaminated) compared to small colonies challenged with non-contaminated fibres only. Conversely, larger colonies moved items regardless of the presence of the spores inside the nest. Colony size qualitatively affected removal of waste items showing a pathogen elicited reaction in small colonies to optimise the reduced workforce, while the removal behaviour in larger colonies revealed to be expressed constitutively.

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis ) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD , gyrB , recA , and rpoB ), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as " A. viscosum " (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named "G19" and "G20." Hence, we propose the strains Rew, Rnw, and Rnr as the members of "G19" and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

Pantoea agglomerans (Pa), a widespread commensal bacterium, has evolved into a host-specific gall-forming pathogen on gypsophila and beet by acquiring a plasmid harbouring a type III secretion system (T3SS) and effectors (T3Es). Pantoea agglomerans pv. gypsophilae (Pag) elicits galls on gypsophila and a hypersensitive response on beet, whereas P. agglomerans pv. betae (Pab) elicits galls on beet and gypsophila. HsvG and HsvB are two paralogous T3Es present in both pathovars and act as host-specific transcription activators on gypsophila and beet, respectively. PthG and PseB are major T3Es that contribute to gall development of Pag and Pab, respectively. To establish the minimal combinations of T3Es that are sufficient to elicit gall symptoms, strains of the nonpathogenic bacteria Pseudomonas fluorescens 55, Pa 3-1, Pa 98 and Escherichia coli, transformed with pHIR11 harbouring a T3SS, and the phytopathogenic bacteria Erwinia amylovora, Dickeya solani and Xanthomonas campestris pv. campestris were transformed with the T3Es hsvG, hsvB, pthG and pseB, either individually or in pairs, and used to infect gypsophila and beet. Strikingly, all the tested nonpathogenic and phytopathogenic bacterial strains harbouring hsvG and pthG incited galls on gypsophila, whereas strains harbouring hsvB and pseB, with the exception of E. coli, incited galls on beet., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)