in-vivo-vitro-infection-potato-roots-with-plant-parasitic-nematodes

<em>In vivo</em> and <em>In vitro</em> Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes

Soil-dwelling plant parasitic nematodes (PPNs) are important potato pests that cause lesions and/or change plant roots structure, leading to reduced crop ...

Universidade de Évora

Faculdade de Ciências da Universidade de Lisboa

Acetone was investigated and found to be an appropriate alternative to Triton X-100 as a solvent of essential oils in bioassays aimed to investigate their effects on pinewood nematode (Bursaphelenchus xylophilus) mortality. Therefore it was used as dilution agent to screen the effectiveness of fifty two essential oils against this pest. Thirteen essential oils were highly effective, resulting in more than 90% pinewood nematode mortality at 2 mg/mL, with six of them resulting in 100% mortality. LC₁₀₀ values ranged between 0.50 mg/mL and 0.83 mg/mL for the essential oils of Origanum vulgare and Satureja montana, respectively. Essential oils were submitted to gas chromatography and gas chromatography-mass spectrometry analysis and their chemical composition established. Data from essential oils with 100% mortality at 2 mg/mL and other essential oils previously found to have LC₁₀₀ ≤ 2 mg/mL was combined, their chemical profiles investigated by correspondences analysis plus automatic classification.

Bioassays against pinewood nematode: assessment of a suitable dilution agent and screening for bioactive essential oils.

Pine Wilt Disease (PWD) is a complex disease integrating three major agents: the pathogenic agent, the pinewood nematode Bursaphelenchus xylophilus; the insect-vector Monochamus spp.; and the host pine tree, Pinus sp. Since the early 80's, the notion that another pathogenic agent, namely bacteria, may play a role in PWD has been gaining traction, however the role of bacteria in PWD is still unknown. The present work supports the possibility that some B. xylophilus-associated bacteria may play a significant role in the development of this disease. This is inferred as a consequence of: (i) the phenotypic characterization of a collection of 35 isolates of B. xylophilus-associated bacteria, in different tests broadly used to test plant pathogenic and plant growth promoting bacteria, and (ii) greenhouse experiments that infer the pathogenicity of these bacteria in maritime pine, Pinus pinaster. The results illustrate the presence of a heterogeneous microbial community associated with B. xylophilus and the traits exhibited by at least, some of these bacteria, appear to be related to PWD symptoms. The inoculation of four specific B. xylophilus-associated bacteria isolates in P. pinaster seedlings resulted in the development of some PWD symptoms suggesting that these bacteria likely play an active role with B. xylophilus in PWD.

Characterization of bacteria associated with pinewood nematode Bursaphelenchus xylophilus.

Pine wilt disease (PWD) has a tremendous impact on worldwide forestlands, both from the environmental and economical viewpoints. Monochamus sp., a xylophagous insect from the Cerambycidae family, plays an important role in dissemination of the pinewood nematode, Bursaphelenchus xylophilus, the primary pathogenic agent of PWD. This study investigates, for the first time, the bacterial communities of Monochamus galloprovincialis collected from Portuguese Pinus pinaster trees and B. xylophilus free, using a metagenomics approach. Overall, our results show that natural bacterial communities of M. galloprovincialis are mainly composed by γ-proteobacteria, Firmicutes and Bacteroidetes, which may be a reflection of insects' feeding diet and habitat characteristics. We also report different bacterial communities' composition in the thorax and abdomen of M. galloprovincialis, with high abundance of Serratia sp. in both. Our results encourage further studies in the possible relationship between bacteria from the insect vector and B. xylophilus.

Characterization of bacterial communities associated with the pine sawyer beetle Monochamus galloprovincialis, the insect vector of the pinewood nematode Bursaphelenchus xylophilus.

Histological Changes in Stems of Pinus sylvestris Seedlings Infected with a Virulent Isolate of the Pinewood Nematode Bursaphelenchus xylophilus.

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, a serious threat to global forest populations of conifers, especially Pinus spp. A time-course study of the essential oils (EOs) of 2-year-old Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris following inoculation with the PWN was performed. The constitutive and nematode inoculation induced EOs components were analyzed at both the wounding or inoculation areas and at the whole plant level. The enantiomeric ratio of optically active main EOs components was also evaluated. External symptoms of infection were observed only in P. pinaster and P. sylvestris 21 and 15 days after inoculation, respectively. The EO composition analysis of uninoculated and unwounded plants revealed the occurrence of chemotypes for P. pinaster, P. halepensis and P. sylvestris, whereas P. pinea showed a homogenous EO composition. When whole plants were evaluated for EO and monoterpene hydrocarbon enantiomeric chemical composition, no relevant qualitative and quantitative differences were found. Instead, EO analysis of inoculated and uninoculated wounded areas revealed an increase of sesquiterpenes and diterpenic compounds, especially in P. pinea and P. halepensis, comparatively to healthy whole plants EOs.

Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris Essential Oils Chemotypes and Monoterpene Hydrocarbon Enantiomers, before and after Inoculation with the Pinewood Nematode Bursaphelenchus xylophilus.

The aim of this study is to understand the biological role of Serratia quinivorans BXF1, a bacterium commonly found associated with Bursaphelenchus xylophilus, the plant parasitic nematode responsible for pine wilt disease. Therefore, we studied strain BXF1 effect in pine wilt disease. We found that strain BXF1 promoted in vitro nematode reproduction. Moreover, the presence of bacteria led to the absence of nematode chitinase gene (Bxcht-1) expression, suggesting an effect for bacterial chitinase in nematode reproduction. Nevertheless, strain BXF1 was unable to colonize the nematode interior, bind to its cuticle with high affinity or protect the nematode from xenobiotic stress. Interestingly, strain BXF1 was able to promote tomato and pine plant-growth, as well as to colonize its interior, thus, acting like a plant-growth promoting endophyte. Consequently, strain BXF1 failed to induce wilting symptoms when inoculated in pine shoot artificial incisions. This bacterium also presented strong antagonistic activities against fungi and bacteria isolated from Pinus pinaster. Our results suggest that B. xylophilus does not possess a strict symbiotic community capable of inducing pine wilt disease symptoms as previously hypothesized. We show that bacteria like BXF1, which possess plant-growth promoting and antagonistic effects, may be opportunistically associated with B. xylophilus, possibly acquired from the bacterial endophytic community of the host pine.

Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus, and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine endophyte with antagonistic effects.

 subsp.  is the causative agent of paratuberculosis. We report here the draft genome sequence of a rare pigmented  subsp.  type C strain, comprising 58 contigs and having a genome size of 4,851,414 bp. The genome will assist in the execution of pigmentation and virulence studies on this mycobacterium.

Draft Genome Sequence of a Rare Pigmented  subsp.  Type C Strain.

Cork oak (Quercus suber) is native to southwest Europe and northwest Africa where it plays a crucial environmental and economical role. To tackle the cork oak production and industrial challenges, advanced research is imperative but dependent on the availability of a sequenced genome. To address this, we produced the first draft version of the cork oak genome. We followed a de novo assembly strategy based on high-throughput sequence data, which generated a draft genome comprising 23,347 scaffolds and 953.3 Mb in size. A total of 79,752 genes and 83,814 transcripts were predicted, including 33,658 high-confidence genes. An InterPro signature assignment was detected for 69,218 transcripts, which represented 82.6% of the total. Validation studies demonstrated the genome assembly and annotation completeness and highlighted the usefulness of the draft genome for read mapping of high-throughput sequence data generated using different protocols. All data generated is available through the public databases where it was deposited, being therefore ready to use by the academic and industry communities working on cork oak and/or related species.

The draft genome sequence of cork oak.

Cork oak is the main cork-producing species worldwide, and plays a significant economic, ecological and social role in the Mediterranean countries, in particular in Portugal and Spain. The ability to produce cork is limited to a few species, hence it must involve specific regulation mechanisms that are unique to these species. However, to date, these mechanisms remain largely understudied, especially with approaches involving the use of high-throughput sequencing technology. In this study, the transcriptome of cork-producing and non-cork-producing  ×  hybrids was analyzed in order to elucidate the differences between the two groups of trees displaying contrasting phenotypes for cork production. The results revealed the presence of a significant number of genes exclusively associated with cork production, in the trees that developed cork. Moreover, several gene ontology subcategories, such as cell wall biogenesis, lipid metabolic processes, metal ion binding and apoplast/cell wall, were only detected in the trees with cork production. These results indicate the existence, at the transcriptome level, of mechanisms that seem to be unique and necessary for cork production, which is an advancement in our knowledge regarding the genetic regulation behind cork formation and production.

Characterization of the cork formation and production transcriptome in  ×  hybrids.

Gene activity is largely controlled by transcriptional regulation through the action of transcription factors and other regulators. QsMYB1 is a member of the R2R3-MYB transcription factor family related to secondary growth, and in particular, with the cork development process. In order to identify the putative gene targets of QsMYB1 across the cork oak genome we developed a ChIP-Seq strategy.

ChIP-Seq reveals that QsMYB1 directly targets genes involved in lignin and suberin biosynthesis pathways in cork oak (Quercus suber).

The impacts of a rapidly changing environment together with the growth in global trade activities has promoted new plant pest pandemic events in forest ecosystems. The pinewood nematode (PWN), , causes strong worldwide economic and ecological impacts. Direct control is performed through trunk injection of powerful nematicides, however many of these (hemi)synthetic compounds have raised ecological and human health concerns for affecting non-target species and accumulating in food products. As sustainable alternatives, essential oils (EOs) have shown very promising results. In this work, available literature on the direct activity of EOs against PWN is reviewed, as a contribution to advance the search for safer and greener biopesticides to be used in sustainable PWD pest management strategies. For the first time, important parameters concerning the bioassays performed, the PWNs bioassayed, and the EOs used are summarized and comparatively analyzed. Ultimately, an overview of the chemical composition of the most active EOs allowed to uncover preliminary guidelines for anti-PWN EO efficiency. The analysis of important information on the volatile phytochemicals composing nematicidal EOs provides a solid basis to engineer sustainable biopesticides capable of controlling the PWN under an integrated pest management framework and contributes to improved forest health.

Phytochemicals as Biopesticides against the Pinewood Nematode : A Review on Essential Oils and Their Volatiles.

Plant-parasitic nematodes (PPNs) are highly damaging pests responsible for heavy losses in worldwide productivity in a significant number of important plant crops. Common pest management strategies rely on the use of synthetic chemical nematicides, which have led to serious concerns regarding their impacts on human health and the environment. Plant natural products, or phytochemicals, can provide a good source of agents for sustainable control of PPNs, due to their intrinsic characteristics such as higher biodegradability, generally low toxicity for mammals, and lower bioaccumulation in the environment. In this work, the nematicidal activity of 39 phytochemicals was determined against the root-lesion nematode (RLN)  using standard direct and indirect contact methodologies. Overall, the RLN was tolerant to the tested phytochemicals at the highest concentration, 2 mg/mL, seldom reaching full mortality. However, high activities were obtained for benzaldehyde, carvacrol, 3-octanol, and thymol, in comparison to other phytochemicals or the synthetic nematicide oxamyl. These phytochemicals were seen to damage nematode internal tissues but not its cuticle shape. Also, the environmental and (eco)toxicological parameters reported for these compounds suggest lower toxicity and higher safety of use than oxamyl. These compounds appear to be good candidates for the development of biopesticides for a more sustainable pest management strategy.

Article	Year
Bioassays against pinewood nematode: assessment of a suitable dilution agent and screening for bioactive essential oils. Molecules (Basel, Switzerland)\| PubMed ID: 23085666	2012
Characterization of bacteria associated with pinewood nematode Bursaphelenchus xylophilus. PloS one\| PubMed ID: 23091599	2012
Characterization of bacterial communities associated with the pine sawyer beetle Monochamus galloprovincialis, the insect vector of the pinewood nematode Bursaphelenchus xylophilus. FEMS microbiology letters\| PubMed ID: 23927049	2013
Histological Changes in Stems of Pinus sylvestris Seedlings Infected with a Virulent Isolate of the Pinewood Nematode Bursaphelenchus xylophilus. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada\| PubMed ID: 26227729	2015
Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris Essential Oils Chemotypes and Monoterpene Hydrocarbon Enantiomers, before and after Inoculation with the Pinewood Nematode ... Chemistry & biodiversity\| PubMed ID: 27623247	2017
Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus, and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine en... Environmental microbiology\| PubMed ID: 27768814	2016
Draft Genome Sequence of a Rare Pigmented subsp. Type C Strain. Genome announcements\| PubMed ID: 29025941	2017
The draft genome sequence of cork oak. Scientific data\| PubMed ID: 29786699	2018
Characterization of the cork formation and production transcriptome in × hybrids. Physiology and molecular biology of plants : an international journal of functional plant biology\| PubMed ID: 30042611	2018
ChIP-Seq reveals that QsMYB1 directly targets genes involved in lignin and suberin biosynthesis pathways in cork oak (Quercus suber). BMC plant biology\| PubMed ID: 30223777	2018
Phytochemicals as Biopesticides against the Pinewood Nematode : A Review on Essential Oils and Their Volatiles. Plants (Basel, Switzerland)\| PubMed ID: 34961085	2021
Nematicidal Activity of Phytochemicals against the Root-Lesion Nematode . Plants (Basel, Switzerland)\| PubMed ID: 38475572	2024

Pedro Barbosa

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