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1.
Microbes Environ ; 35(1)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101840

RESUMO

The genome of Streptomyces scabies, the predominant causal agent of potato common scab, encodes a potential cutinase, the protein Sub1, which was previously shown to be specifically induced in the presence of suberin. The sub1 gene was expressed in Escherichia coli and the recombinant protein Sub1 was purified and characterized. The enzyme was shown to be versatile because it hydrolyzes a number of natural and synthetic substrates. Sub1 hydrolyzed p-nitrophenyl esters, with the hydrolysis of those harboring short carbon chains being the most effective. The Vmax and Km values of Sub1 for p-nitrophenyl butyrate were 2.36 mol g-1 min-1 and 5.7 10-4 M, respectively. Sub1 hydrolyzed the recalcitrant polymers cutin and suberin because the release of fatty acids from these substrates was observed following the incubation of the enzyme with these polymers. Furthermore, the hydrolyzing activity of the esterase Sub1 on the synthetic polymer polyethylene terephthalate (PET) was demonstrated by the release of terephthalic acid (TA). Sub1 activity on PET was markedly enhanced by the addition of Triton and was shown to be stable at 37°C for at least 20 d.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Doenças das Plantas/microbiologia , Polímeros/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Ácidos Graxos/metabolismo , Hidrólise , Ácidos Ftálicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solanum tuberosum/microbiologia , Streptomyces/genética
2.
World J Microbiol Biotechnol ; 36(1): 18, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31912374

RESUMO

In this paper, we report the antimicrobial activity of AMEP412 (a protein elicitor from Bacillus subtilis) against Streptomyces scabiei, which is the potato common scab pathogen. The purified protein samples showed an obvious inhibition zone on an S. scabiei agar plate, and the minimum inhibition concentration detected was 50 µg mL-1. The fluorescence localization assay revealed that AMEP412 could bind to aerial mycelia and spores. The stability test showed that AMEP412 was stable at 60 °C for 30 min and in pH values from 5.0 to 10.0. Its antimicrobial activity was not sensitive to metal cations. However, its activity declined by 23% when treated with Proteinase K, and was completely abrogated with Tween 80 treatment. Three antimicrobial peptides (GS21, GY20 and GY23) were identified from AMEP412, which further verified its antimicrobial activity. This research reveals the antimicrobial function of AMEP412, which not only enriches the function of the protein elicitor, but also provides a candidate for the biocontrol of potato common scab.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/farmacologia , Streptomyces/efeitos dos fármacos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Micélio/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Solanum tuberosum/microbiologia , Esporos Bacterianos/efeitos dos fármacos , Streptomyces/crescimento & desenvolvimento
3.
PLoS One ; 15(1): e0221604, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31961875

RESUMO

Phytophthora infestans (Mont.) de Bary, a hemibiotrophic oomycete, has caused severe epidemics of late blight in tomato and potato crops around the world since the Irish Potato Famine in the 1840s. Breeding of late blight resistant cultivars is one of the most effective strategies to overcome this disruptive disease. However, P. infestans is able to break down host resistance and acquire resistance to various fungicides, possibly because of the existence of high genetic variability among P. infestans isolates via sexual and asexual reproduction. Therefore, to manage this disease, it is important to understand the genetic divergence of P. infestans isolates. In this study, we analyzed the genomes of P. infestans isolates collected from Egypt and Japan using various molecular approaches including the mating type assay and genotyping simple sequence repeats, mitochondria DNA, and effector genes. We also analyzed genome-wide single nucleotide polymorphisms using double-digest restriction-site associated DNA sequencing and whole genome resequencing (WGRS). The isolates were classified adequately using high-resolution genome-wide approaches. Moreover, these analyses revealed new clusters of P. infestans isolates in the Egyptian population. Monitoring the genetic divergence of P. infestans isolates as well as breeding of resistant cultivars would facilitate the elimination of the late blight disease.


Assuntos
Genes Fúngicos Tipo Acasalamento/genética , Sequenciamento de Nucleotídeos em Larga Escala , Phytophthora infestans/genética , Doenças das Plantas/microbiologia , DNA Mitocondrial/genética , Fungicidas Industriais/farmacologia , Genótipo , Lycopersicon esculentum/microbiologia , Repetições de Microssatélites/genética , Phytophthora infestans/crescimento & desenvolvimento , Doenças das Plantas/genética , Análise de Sequência de DNA , Solanum tuberosum/microbiologia
4.
Plant Dis ; 104(1): 211-221, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31765279

RESUMO

Phytophthora infestans is the causal agent of late blight disease of potatoes and tomatoes. This disease causes devastating economic losses each year, and control is mainly achieved by the use of fungicides. Unfortunately, populations of P. infestans resistant to fungicides have been documented. Furthermore, studies have reported that sensitive isolates to the phenylamide fungicide, mefenoxam, become less sensitive in vitro after a single passage through sublethal concentrations of fungicide-amended medium. The first objective of this study was to investigate if isolates of P. infestans are capable of acquiring resistance to two additional systemic fungicides, fluopicolide (benzamide) and cymoxanil (cyanoacetamide-oxime). In contrast to the situation with mefenoxam, exposure of isolates to sublethal concentrations of fluopicolide and cymoxanil did not induce reduced sensitivity to these two fungicides. The second objective was to assess if reduced sensitivity to mefenoxam could occur in naturally sensitive isolates of other Phytophthora species and of Phytopythium sp., another oomycete plant pathogen. All Phytophthora spp. assessed (P. infestans, P. betacei, and P. pseudocryptogea) as well as Phytopythium sp. acquired resistance to mefenoxam after previous exposure through medium containing 1 µg ml-1 of mefenoxam. Interestingly, isolate 66 of Phytopythium sp. and the isolate of P. pseudocryptogea tested do not seem to be acquiring resistance to mefenoxam after exposure to medium containing 5 µg ml-1 of this fungicide. The tested isolates of P. palmivora and P. cinnamomi were extremely sensitive to mefenoxam, and thus it was not possible to perform a second transfer to access acquisition of resistance to this fungicide.


Assuntos
Alanina/análogos & derivados , Farmacorresistência Fúngica , Phytophthora infestans , Alanina/farmacologia , Fungicidas Industriais/farmacologia , Phytophthora infestans/efeitos dos fármacos , Solanum tuberosum/microbiologia
5.
Plant Biol (Stuttg) ; 22(1): 81-89, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31539455

RESUMO

Potato common scab is a worldwide disease mainly caused by Streptomyces scabiei. It seriously affects potato crops by decreasing tuber quality. Essential oils (EO) are natural products with recognised antimicrobial properties. In this research, the antibacterial activities of thyme, oregano, suico and mint EO against S. scabiei were analysed. Infected tubers and soil samples were used for bacterial isolation; the obtained isolates were genetically identified. The chemical composition of the EO was determined by GC-MS. The broth microdilution method was used to analyse antibacterial properties of EO. Thirty-one bacterial isolates were obtained. The isolate chosen for antibacterial assays was morpho-physiologically and genetically identified as S. scabiei. Thyme EO was mainly composed of thymol and o-cymene; suico EO of dihydrotagetone, trans-tagetone and verbenone; oregano EO of trans-sabinene hydrate, thymol and É£-terpinene; and mint EO of menthone and menthol. All the EO tested were effective against S. scabiei, but thyme and suico EO were the most successful, with a minimum inhibitory concentration of 0.068 g·l-1 and 0.147 g·l-1 , respectively, and a minimum bactericidal concentration of 0.137 g·l-1 and 0.147 g·l-1 , respectively. Scanning electron microscopy showed similar damage caused by both thyme and suico EO to the bacterial envelope. Total phenolic content of EO was not related to their antibacterial activity. Thyme and suico EO are effective antibacterial agents against S. scabiei, impeding bacterial viability and disturbing the bacterial cell envelope. These EO are promising tools for control of potato common scab.


Assuntos
Óleos Voláteis , Solanum tuberosum , Streptomyces , Anti-Infecciosos/farmacologia , Mentha/química , Testes de Sensibilidade Microbiana , Óleos Voláteis/farmacologia , Origanum/química , Doenças das Plantas/prevenção & controle , Solanum tuberosum/microbiologia , Streptomyces/efeitos dos fármacos , Thymus (Planta)/química
6.
World J Microbiol Biotechnol ; 35(12): 195, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31784916

RESUMO

The search for effective plant-growth-promoting strains of rhizospheric bacteria that would ensure the resistance of plant-microbial associations to environmental stressors is essential for the design of environmentally friendly agrobiotechnologies. We investigated the interaction of potato (cv. Nevsky) microplants with the plant-growth-promoting bacteria Azospirillum brasilense Sp245 and Ochrobactrum cytisi IPA7.2 under osmotic stress in vitro. The bacteria improved the physiological and biochemical variables of the microplants, significantly increasing shoot length and root number (1.3-fold, on average). Inoculation also led a more effective recovery of the plants after stress. During repair, inoculation contributed to a decreased leaf content of malonic dialdehyde. With A. brasilense Sp245, the decrease was 1.75-fold; with O. cytisi IPA7.2, it was 1.4-fold. During repair, the shoot length, node number, and root number of the inoculated plants were greater than the control values by an average of 1.3-fold with A. brasilense Sp245 and by an average of 1.6-fold with O. cytisi IPA7.2. O. cytisi IPA7.2, previously isolated from the potato rhizosphere, protected the physiological and biochemical processes in the plants under stress and repair better than did A. brasilense Sp245. Specifically, root weight increased fivefold during repair, as compared to the noninoculated plants, while chlorophyll a content remained at the level found in the nonstressed controls. The results indicate that these bacteria can be used as components of biofertilizers. A. brasilense Sp245 has favorable prospects for use in temperate latitudes, whereas O. cytisi IPA7.2 can be successfully used in saline and drought-stressed environments.


Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Pressão Osmótica , Desenvolvimento Vegetal , Solanum tuberosum/microbiologia , Estresse Fisiológico/fisiologia , Azospirillum brasilense/fisiologia , Clorofila A , Contagem de Colônia Microbiana , Secas , Malonatos , Ochrobactrum/fisiologia , Folhas de Planta , Raízes de Plantas/microbiologia , Brotos de Planta , Rizosfera
7.
World J Microbiol Biotechnol ; 35(12): 181, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31728652

RESUMO

Root-associated fungi and bacteria play a pivotal role in the plant-soil ecosystem by influencing both plant growth and immunity. The aim of this study was to unravel the biodiversity of the bacterial and fungal rhizosphere (RS) and rhizoplane (RP) microbiota of Zhukovskij rannij potato (Solanum tuberosum L.) cultivar growing in the Alfisol of Tatarstan, Russia. To assess the structure and diversity of microbial communities, we employed the 16S rRNA and internal transcribed spacer gene library technique. Overall, sequence analysis showed the presence of 3982 bacterial and 188 fungal operational taxonomic units (OTUs) in the RP, and 6018 bacterial and 320 fungal OTUs for in the RS. Comparison between microbial community structures in the RS and RP showed significant differences between these compartments. Biodiversity was higher in the RS than in the RP. Although members of Proteobacteria (RS-59.1 ± 4.9%; RP-54.5 ± 9.2%), Bacteroidetes (RS-23.19 ± 10.2%; RP-34.52 ± 10.4%) and Actinobacteria (RS-11.55 ± 4.9%; RP-7.7 ± 5.1%) were the three most dominant phyla, accounting for 94-98% of all bacterial taxa in both compartments, notable variations were observed in the primary dominance of classes and genera in RS and RP samples. In addition, our results demonstrated that the potato rhizoplane was significantly enriched with the genera Flavobacterium, Pseudomonas, Acinetobacter and other potentially beneficial bacteria. The fungal community was predominantly inhabited by members of the Ascomycota phylum (RS-81.4 ± 8.1%; RP-81.7 ± 5.7%), among which the genera Fusarium (RS-10.34 ± 3.41%; RP-9.96 ± 4.79%), Monographella (RS-7.66 ± 4.43%; RP-9.91 ± 5.87%), Verticillium (RS-4.6 ± 1.43%; RP-8.27 ± 3.63%) and Chaetomium (RS-4.95 ± 2.07%; RP-8.33 ± 4.93%) were particularly abundant. Interestingly, potato rhizoplane was significantly enriched with potentially useful fungal genera, such as Mortierella and Metacordiceps. A comparative analysis revealed that the abundance of Fusarium (a cosmopolitan plant pathogen) varied significantly depending on rotation variants, indicating a possible control of phytopathogenic fungi via management-induced shifts through crop rotational methods. Analysis of the core microbiome of bacterial and fungal community structure showed that the formation of bacterial microbiota in the rhizosphere and rhizoplane is dependent on the host plant.


Assuntos
Bactérias/classificação , Biodiversidade , Fungos/classificação , Microbiota/fisiologia , Raízes de Plantas/microbiologia , Solo/química , Solanum tuberosum/microbiologia , Bactérias/genética , Fungos/genética , Microbiota/genética , Filogenia , Desenvolvimento Vegetal , RNA Ribossômico 16S/genética , Rizosfera , Federação Russa , Microbiologia do Solo , Solanum tuberosum/crescimento & desenvolvimento
8.
Plant Dis ; 103(12): 3150-3153, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31596689

RESUMO

The objective of this research was to test the hypothesis that Verticillium isaacii causes diseases of sunflower and potato plants. Two sunflower genotypes and one potato cultivar were inoculated with five V. isaacii isolates and three pathogenic V. dahliae isolates. Biomass, disease expression, and stem colonization were quantified. Overt wilt symptoms were observed on both sunflower genotypes and potato plants inoculated with a subset of the V. isaacii isolates. Biomass of both sunflower genotypes was not affected by V. isaacii infection. Tuber yields either decreased in response to infection by one V. isaacii isolate or were not affected by infection. Stems of sunflower and potato plants were infected by at least four of the five V. isaacii isolates. A new disease of sunflower and potato is documented. Evidence that V. isaacii exhibits different lifestyles including pathogenicity and endophytism is presented. Finally, this research documents variation in fungal lifestyles that can exist in samples from a single field.


Assuntos
Helianthus , Solanum tuberosum , Verticillium , Endófitos/fisiologia , Genes Fúngicos/genética , Genótipo , Helianthus/microbiologia , Especificidade de Hospedeiro , Solanum tuberosum/microbiologia , Verticillium/genética , Verticillium/fisiologia , Washington
9.
Plant Dis ; 103(11): 2821-2824, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31509493

RESUMO

Early blight is an economically important foliar disease of potato in the United States. Because of the lack of resistant potato cultivars, fungicides are applied extensively to obtain adequate control. To manage early blight, standard protectant fungicides and single-site mode-of-action "specialty" fungicides are applied either alone or incorporated into a fungicide rotation program. Control efficacy at two crop growth stages (tuber initiation/early bulking and late bulking/tuber maturation) and the overall tuber yield response to standard and specialty fungicides were assessed using network metaanalytic models. Control efficacy of fungicides ranged from moderate to very high (>30 to 75%) compared with the nontreated control. For both potato growth stages, specialty fungicides performed better than standard protectant fungicides. Furthermore, control efficacy of both fungicides was higher (3 to 9%) at late bulking and tuber maturation when compared with early bulking crop growth stage. Specialty fungicide programs increased overall tuber yields by 4 and 9% over standard fungicides and nontreated control, respectively. Based on the results, more precise fungicide use recommendations and fungicide programs can be developed for early blight management.


Assuntos
Fungicidas Industriais , Doenças das Plantas , Solanum tuberosum , Fungicidas Industriais/normas , Doenças das Plantas/prevenção & controle , Solanum tuberosum/microbiologia , Estados Unidos
10.
PLoS Pathog ; 15(8): e1007703, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381590

RESUMO

Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen. Infected plants lose tissue integrity and functional immunity as a result, exposing the nutrient rich, decayed tissues to the environment. One challenge for the necrotrophs to successfully cause secondary infection (infection spread from an initially infected plant to the nearby uninfected plants) is to effectively utilize nutrients released from hosts towards building up a large population before other saprophytes come. In this study, we observed that the necrotrophic pathogen Dickeya dadantii exhibited heterogeneity in bacterial cell length in an isogenic population during infection of potato tuber. While some cells were regular rod-shape (<10µm), the rest elongated into filamentous cells (>10µm). Short cells tended to occur at the interface of healthy and diseased tissues, during the early stage of infection when active attacking and killing is occurring, while filamentous cells tended to form at a later stage of infection. Short cells expressed all necessary virulence factors and motility, whereas filamentous cells did not engage in virulence, were non-mobile and more sensitive to environmental stress. However, compared to the short cells, the filamentous cells displayed upregulated metabolic genes and increased growth, which may benefit the pathogens to build up a large population necessary for the secondary infection. The segregation of the two subpopulations was dependent on differential production of the alarmone guanosine tetraphosphate (ppGpp). When exposed to fresh tuber tissues or freestanding water, filamentous cells quickly transformed to short virulent cells. The pathogen adaptation of cell length heterogeneity identified in this study presents a model for how some necrotrophs balance virulence and vegetative growth to maximize fitness during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/química , Guanosina Tetrafosfato/metabolismo , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Enterobacteriaceae/metabolismo , Enterobacteriaceae/patogenicidade , Regulação Bacteriana da Expressão Gênica , Doenças das Plantas/imunologia , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/metabolismo , Virulência , Fatores de Virulência/genética
11.
Plant Dis ; 103(11): 2893-2902, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31436473

RESUMO

Uniqprimer, a software pipeline developed in Python, was deployed as a user-friendly internet tool in Rice Galaxy for comparative genome analyses to design primer sets for PCRassays capable of detecting target bacterial taxa. The pipeline was trialed with Dickeya dianthicola, a destructive broad-host-range bacterial pathogen found in most potato-growing regions. Dickeya is a highly variable genus, and some primers available to detect this genus and species exhibit common diagnostic failures. Upon uploading a selection of target and nontarget genomes, six primer sets were rapidly identified with Uniqprimer, of which two were specific and sensitive when tested with D. dianthicola. The remaining four amplified a minority of the nontarget strains tested. The two promising candidate primer sets were trialed with DNA isolated from 116 field samples from across the United States that were previously submitted for testing. D. dianthicola was detected in 41 samples, demonstrating the applicability of our detection primers and suggesting widespread occurrence of D. dianthicola in North America.


Assuntos
Agricultura , Técnicas Bacteriológicas , Primers do DNA , Enterobacteriaceae , Solanum tuberosum , Agricultura/métodos , Técnicas Bacteriológicas/métodos , Primers do DNA/genética , Enterobacteriaceae/genética , América do Norte , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia
12.
Genes (Basel) ; 10(9)2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466418

RESUMO

Fusarium oxysporum is the most important pathogen of potatoes which causes post-harvest destructive losses and deteriorates the market value of potato tubers worldwide. Here, F. oxysporum was used as a host pathogen model system and it was revealed that autophagy plays a vital role as a regulator in the morphology, cellular growth, development, as well as the pathogenicity of F. oxysporum. Previous studies based upon identification of the gene responsible for encoding the autophagy pathway components from F. oxysporum have shown putative orthologs of 16 core autophagy related-ATG genes of yeast in the genome database which were autophagy-related and comprised of ubiquitin-like protein atg3. This study elucidates the molecular mechanism of the autophagy-related gene Foatg3 in F. oxysporum. A deletion (∆) mutants of F. oxysporum (Foatg3∆) was generated to evaluate nuclear dynamics. As compared to wild type and Foatg3 overexpression (OE) strains, Foatg3∆ strains failed to show positive MDC (monodansylcadaverine) staining which revealed that Foatg3 is compulsory for autophagy in F. oxysporum. A significant reduction in conidiation and hyphal growth was shown by the Foatg3∆ strains resulting in loss of virulence on potato tubers. The hyphae of Foatg3∆ mutants contained two or more nuclei within one hyphal compartment while wild type hyphae were composed of uninucleate hyphal compartments. Our findings reveal that the vital significance of Foatg3 as a key target in controlling the dry rot disease in root crops and potato tubers at the postharvest stage has immense potential of disease control and yield enhancement.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Proteínas Fúngicas/genética , Fusarium/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Deleção de Genes , Hifas/genética , Hifas/crescimento & desenvolvimento , Solanum tuberosum/microbiologia , Virulência/genética
13.
Plant Dis ; 103(10): 2587-2591, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432751

RESUMO

'Candidatus Liberibacter solanacearum' (Lso) is an uncultured, phloem-associated bacterium causing a severe tuber disease in potato called zebra chip (ZC). Seven haplotypes of Lso have been described in different hosts, with haplotypes A and B found associated with infections in potato and tomato. In the field, Lso is transmitted by the potato psyllid (Bactericera cockerelli), and between 2011 and 2015, a significant change in Lso haplotype prevalence was previously reported in Idaho: from exclusively A haplotype found in tested psyllids in 2012 to mainly B haplotype found in collected psyllids in 2015. However, prevalence of Lso haplotypes in Idaho was not analyzed in potato tubers exhibiting symptoms of ZC. To fill in this knowledge gap, prevalence of Lso haplotypes was investigated in potato tubers harvested in southern Idaho between 2012 and 2018, and it was found to change from exclusively A haplotype in the 2012 season to an almost equal A and B haplotype distribution during the 2016 season. During the same period, haplotype distribution of Lso in psyllid vectors collected using yellow sticky traps also changed, but in psyllids, the shift from A haplotype of Lso to B haplotype was complete, with no A haplotype detected in 2016 to 2018. The changes in the haplotype prevalence of the Lso circulating in potato fields in southern Idaho may be, among other factors, responsible for a decrease in the ZC incidence in Idaho potato fields between an outbreak of the disease in 2012 and a very low level of ZC afterward.


Assuntos
Hemípteros , Rhizobiaceae , Solanum tuberosum , Animais , Haplótipos , Hemípteros/microbiologia , Idaho , Rhizobiaceae/fisiologia , Solanum tuberosum/microbiologia
14.
BMC Res Notes ; 12(1): 457, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31345264

RESUMO

OBJECTIVE: GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). We examined the ability of the GAANTRY Agrobacterium rhizogenes ArPORT1 '10-stack' strain to generate transgenic potato plants. RESULTS: The 28.5 kilobase 10-stack T-DNA, was introduced into Lenape potato plants with a 32% transformation efficiency. Molecular and phenotypic characterization confirmed that six of the seven tested independent transgenic lines carried the entire desired construct, demonstrating that the GAANTRY 10-stack strain can be used can be used in a tissue culture-based callus transformation method to efficiently generate transgenic potato plants. Analysis using droplet digital PCR showed that most of the characterized events carry one or two copies of the 10-stack transgenes and that 'backbone' DNA from outside of the T-DNA was absent in the transgenic plants. These results demonstrate that the GAANTRY system efficiently generates high quality transgenic potato plants with a large construct of stacked transgenes.


Assuntos
Agrobacterium/genética , DNA Bacteriano/genética , Técnicas de Transferência de Genes , Plasmídeos/metabolismo , Solanum tuberosum/genética , Transgenes , Agrobacterium/metabolismo , DNA Bacteriano/metabolismo , Dosagem de Genes , Expressão Gênica , Genes Reporter , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fenótipo , Folhas de Planta/genética , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Plasmídeos/química , Reação em Cadeia da Polimerase/métodos , Solanum tuberosum/microbiologia
15.
Mar Drugs ; 17(7)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261836

RESUMO

N-Acylhomoserine lactonase degrades the lactone ring of N-acylhomoserine lactones (AHLs) and has been widely suggested as a promising candidate for use in bacterial disease control. While a number of AHL lactonases have been characterized, none of them has been developed as a commercially available enzymatic product for in vitro AHL quenching due to their low stability. In this study, a highly stable AHL lactonase (AhlX) was identified and isolated from the marine bacterium Salinicola salaria MCCC1A01339. AhlX is encoded by a 768-bp gene and has a predicted molecular mass of 29 kDa. The enzyme retained approximately 97% activity after incubating at 25 °C for 12 days and ~100% activity after incubating at 60 °C for 2 h. Furthermore, AhlX exhibited a high salt tolerance, retaining approximately 60% of its activity observed in the presence of 25% NaCl. In addition, an AhlX powder made by an industrial spray-drying process attenuated Erwinia carotovora infection. These results suggest that AhlX has great potential for use as an in vitro preventive and therapeutic agent for bacterial diseases.


Assuntos
Antibacterianos/farmacologia , Organismos Aquáticos/enzimologia , Proteínas de Bactérias/farmacologia , Hidrolases de Éster Carboxílico/farmacologia , Halomonadaceae/enzimologia , Acil-Butirolactonas/química , Antibacterianos/química , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Biotecnologia , Brassica rapa/microbiologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/isolamento & purificação , Ensaios Enzimáticos , Estabilidade Enzimática , Pectobacterium carotovorum/efeitos dos fármacos , Pectobacterium carotovorum/fisiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Percepção de Quorum/efeitos dos fármacos , Solanum tuberosum/microbiologia , Temperatura
16.
Phytopathology ; 109(11): 1922-1931, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31272278

RESUMO

Bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is a serious threat to potato production in Uganda. However, little is known about the extent of the disease and the type of the pathogen strains involved. A nationwide survey was conducted to study BW prevalence and incidence in potato, and potato tuber and stem samples of potential alternative hosts were collected for pathogen isolation. DNA was extracted from pure cultures for genetic diversity studies. The pathogen was phylotyped by multiplex PCR; then, a subset of isolates was typed at sequevar level. Isolates of the same sequevar were then haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. BW prevalence and incidence in potato farms were 81.4 and 1.7%, respectively. Three RSSC phylotypes were identified, with the majority of the strains belonging to Phylotype II (80%) followed by Phylotype I (18.5%) and III (1.5%). Phylotype I strains belonged to Sequevar 31, and Phylotype II strains belonged to Sequevar 1. Potato-associated Phylotype II Sequevar 1 strains were more diverse (27 TRST haplotypes) than nonpotato Phylotype I (5 TRST haplotypes). Mapping of TRST haplotypes revealed that three TRST haplotypes of Phylotype II Sequevar 1 strains play an important epidemiological role in BW of potato in Uganda being disseminated via latently infected seed.[Formula: see text]Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.


Assuntos
Epidemiologia Molecular , Ralstonia solanacearum , Solanum tuberosum , Tipagem Molecular , Filogenia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética , Solanum tuberosum/microbiologia , Uganda
17.
Microbiology ; 165(10): 1025-1040, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31162023

RESUMO

Gram-positive Actinobacteria from the genus Streptomyces are best known for their morphological complexity and for their ability to produce numerous bioactive specialized metabolites with useful applications in human and veterinary medicine and in agriculture. In contrast, the ability to infect living plant tissues and to cause diseases of root and tuber crops such as potato common scab (CS) is a rare attribute among members of this genus. Research on the virulence mechanisms of plant-pathogenic Streptomyces spp. has revealed the importance of the thaxtomin phytotoxins as key pathogenicity determinants produced by several species. In addition, other phytotoxic specialized metabolites may contribute to the development or severity of disease caused by Streptomyces spp., along with the production of phytohormones and secreted proteins. A thorough understanding of the molecular mechanisms of plant pathogenicity will enable the development of better management procedures for controlling CS and other plant diseases caused by the Streptomyces.


Assuntos
Doenças das Plantas/microbiologia , Streptomyces/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Reguladores de Crescimento de Planta/química , Reguladores de Crescimento de Planta/genética , Reguladores de Crescimento de Planta/metabolismo , Tubérculos/microbiologia , Solanum tuberosum/microbiologia , Streptomyces/genética , Streptomyces/metabolismo , Virulência
18.
Microb Pathog ; 135: 103604, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31247257

RESUMO

The present study describes the possibility of using some essential oils and monoterpens as bioagents against the growth of Ralstonia solanacearum, a causal bacterium of potato brown rot disease. Eight isolates of the bacterium were recovered from infected potato tubers, showing typical symptoms of the disease, Isolates were identified as R.solanacearum phylotype II, based on biochemical and physiological characteristics, as well as, at the molecular level through PCR analysis. Three essential oils extracted from Corymbia citriodora (leaves), Cupressus sempervirens (aerial parts), and Lantana camara (aerial parts) were evaluated for their antibacterial activity against eight isolates of R. solanacearum phylotype II. Results demonstrated that L. camara essential oil (concentration 5000 µg/mL) had the highest effects against the RsMo2, RsSc1 and Rs48, with inhibition zone (IZ) values of 17.33, 16.33, and 17.50 mm, respectively, also against Rs2 (IZ 14.33 mm), and RsIs2 (IZ 16 mm). C. citriodora oil showed the highest activity against RsBe2 (IZ 14 mm), RsFr4 (IZ 13.66 mm) and RsNe1 (IZ 13.66 mm). Gas Chromatography-Mass Spectrometry (GC-MS-FID) analyzed the chemical composition of these essential oils. It was proved that L. camara leaves contains mainly trans-caryophyllene (16.24%) and α-humulene (9.55%), in C. citriodora oil were α-citronellal (56.55%), α-citronellol (14.89%), and citronellol acetate (13.04%), and in Cup. sempervirens aerial parts were cedrol (22.17%), and Δ3-carene (18.59%). Five monoterpenes were evaluated against the most resistance Ralstonia isolate RsFr5 to the three studied essential oils and found that limonene had the highest effect against it compared with the lowest thymol. The results proved the strong bio effects of the essential oil from L. camara leaves as a natural product contained monoterpenes that can inhibit the growth of tested R. solanacearum phylotype II isolates.


Assuntos
Antibacterianos/farmacologia , Monoterpenos/farmacologia , Óleos Voláteis/farmacologia , Extratos Vegetais/farmacologia , Ralstonia solanacearum/efeitos dos fármacos , Solanum tuberosum/microbiologia , Aldeídos/isolamento & purificação , Aldeídos/farmacologia , Antibacterianos/isolamento & purificação , Cupressus/química , DNA Bacteriano/análise , Cromatografia Gasosa-Espectrometria de Massas , Lantana/química , Testes de Sensibilidade Microbiana , Monoterpenos/isolamento & purificação , Myrtaceae/química , Octanóis/isolamento & purificação , Octanóis/farmacologia , Óleos Voláteis/isolamento & purificação , Doenças das Plantas/microbiologia , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Ralstonia solanacearum/crescimento & desenvolvimento , Ralstonia solanacearum/isolamento & purificação , Ralstonia solanacearum/fisiologia , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia
19.
Plant Dis ; 103(8): 2033-2040, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31232655

RESUMO

Quinone outside inhibitor (QoI) fungicides have been an important class in managing potato early blight caused by Alternaria solani and brown spot caused by A. alternata. Because of the single-site mode of action character of QoI fungicides, which are relied on for management of diseases in Wisconsin, and the abundant asexual conidia production of the Alternaria species, pathogen isolates with QoI resistance have been detected after just a few years of QoI fungicide usage in commercial production fields. Resistance to QoIs has been attributed to amino acid substitutions F129L and G143A in cytochrome b of A. solani and A. alternata, respectively, as a result of point mutations. The aim of this study was to assess Alternaria populations in Wisconsin for QoI resistance before and after fungicide applications in order to evaluate resistance stability. A TaqMan single nucleotide polymorphism genotyping assay was designed based on the sequences of the cytochrome b gene from Alternaria isolates collected in Wisconsin to profile QoI resistance in Alternaria populations as well as to explore factors that may influence frequency of QoI resistance in the pathogen populations. This assay successfully identified the mutations conferring QoI resistance in isolates collected from four locations each year from 2015 to 2017. During the course of this study, the frequency of A. solani isolates with the F129L mutation was consistently high and showed primarily the TTA mutation type. The frequency of A. alternata isolates with the G143A mutation started relatively low and increased at the end of the production season in each year (P = 0.0109, P = 0.2083, and P = 0.0159). A potato field managed without use of QoI fungicides showed a significantly lower (P < 0.05) frequency of A. alternata isolates carrying G143A than conventionally managed potato fields. The overall frequency of A. alternata isolates carrying G143A in the four locations was similar over the 3 years (P = 0.2971). The QoI resistance characteristics of the isolates were stable even when QoI selection pressure was removed for at least five subculture transfers, and the mutation types of codons 129 and 143 in the cytochrome b gene in A. solani and A. alternata, respectively, remained the same. This indicated that the application of QoIs in the field is not the sole factor responsible for the variation of the frequency of QoI resistance in the pathogen populations.


Assuntos
Alternaria , Farmacorresistência Fúngica , Fungicidas Industriais , Solanum tuberosum , Alternaria/efeitos dos fármacos , Alternaria/fisiologia , Fungicidas Industriais/farmacologia , Solanum tuberosum/microbiologia , Wisconsin
20.
PLoS One ; 14(6): e0218868, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31233546

RESUMO

Destructive maceration, a wide host range, and longevity in non-plant substrates has established Dickeya dianthicola (blackleg of potato) as a significant threat to potato industries worldwide. To protect these businesses, a specific and sensitive point-of-care D. dianthicola detection tool is necessary. We have developed a loop-mediated isothermal amplification (LAMP) assay for specific, sensitive, and rapid detection of D. dianthicola, which can be streamlined for point-of-care use. The developed LAMP assay targets a unique gene, alcohol dehydrogenase, of D. dianthicola. Assay specificity was assessed using strains present in inclusivity (16 D. dianthicola strains) and exclusivity panels (56 closely related, potato pathogenic, and other bacterial strains). Amplification with strains of inclusivity panel occurred, and cross-reactivity with non-target DNA was not observed. The limit of detection (LOD) was 10 CFU/ml when dilutions were made before isolating the genomic DNA; however, LOD was determined as 1 pg using 10-fold serially diluted D. dianthicola genomic DNA. Similar LOD of 1 pg was observed when serially diluted target genomic DNA was mixed with host genomic DNA. LOD (1 pg) was also calculated with 10-fold serially diluted synthetic DNA fragments containing primer target sites. Naturally and artificially inoculated plant samples were used for field adaptability tests with the field-deployable Optigene Plant Material Lysis Kit and a heat block (65°C); the results were obtained within 20 minutes. Despite the lack of method precision, no false positives or false negatives were observed. Therefore, with prepared reactions and a steady heat source, this assay can be used for rapid point-of-care detection, which is imperative for quarantine, eradication, disease management, and border protection.


Assuntos
Álcool Desidrogenase/genética , Gammaproteobacteria/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Solanum tuberosum/microbiologia , Gammaproteobacteria/isolamento & purificação , Limite de Detecção , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Sensibilidade e Especificidade , Fatores de Tempo
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