39 research outputs found
Molecular characterization of Portuguese populations of the pinewood nematode Bursaphelenchus xylophilus using cytochrome b and cellulase genes
Bursaphelenchus xylophilus is the causal agent of pine wilt disease and a
worldwide pest with high economic impact. Since its first diagnosis in Portugal
in 1999, it has been subjected to quarantine measures with impact on forest
health and ecosystem stability, significantly affecting international trade of wood
products. The disease was detected in the north and centre of continental
Portugal and, since 2008, the whole country has been considered an affected
area. Recently, it was detected in Madeira Island. In order to avoid new
outbreaks, it has become of major importance to understand the patterns of
spread, introduction points and to characterize the new populations from
continental Portugal and Madeira Island. Mitochondrial cytochrome b (cytb) and
parasitic cellulase gene sequences were used to evaluate the genetic
relationships among isolates that could indicate possible origins of the new
outbreaks. Portuguese isolates were compared with isolates from USA, China,
Japan and South Korea, in order to investigate possible infection pathways and
disease spread patterns in Portugal. Phylogenetic trees based on both genes
show that Portuguese isolates group with Asian isolates. Isolates from USA are
in a separate position in both gene trees. However, the phylogenetic tree based
on the cellulase gene sequences shows higher differentiation among Portuguese
isolates than that of cytb. These results agree with those previously obtained
using inter-simple sequence repeats (ISSR). This was the first study to use cytb
and cellulase genes to characterize pinewood nematode (PWN) populations.
This study suggests that cellulase is a better marker than cytb to study genetic
diversity in B. xylophilus
Genetic diversity of entomopathogenic nematodes (Nematoda: Steinernematidae and Heterorhabditidae) and the nematode Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) from continental Portugal
“Diversidade genética dos nemátodes entomopatogénicos
(Nematoda: Steinernematidae e Heterorhabditidae) e do
nemátode Bursaphelenchus xylophilus (Nematoda:
Aphelenchoididade) em Portugal continental”
Os nematodes entomopatogénicos são utilizados como agentes de controlo
biológico. Para compreender a sua diversidade, foi realizada uma prospecção
em Portugal. Cinco espécies, nomeadamente Steinernema feltiae, S.
intermedium, S. kraussei, Steinernema sp. e Heterorhabditis bacteriophora
foram identificadas. As sequências de ITS, região D2D3 do 28S rRNA, COXI e
cytb foram utilizadas para estudar a diversidade genética das duas espécies
mais abundantes, S. feltiae and H. bacteriophora, não tendo sido encontradas
diferenças significativas entre isolados.
O nemátode da madeira do pinheiro, Bursaphelenchus xylophilus, provoca
doença nos pinheiros tendo sido detectada pela primeira vez na Europa e em
Portugal em 1999. Para avaliar a diversidade genética dos isolados
Portugueses e identificar o padrão de propagação da doença, foram utilizadas
a sequência da região IGS do 5.8S rRNA, e os genes cytb e cellulase,
combinados com os padrões ISSR. Os padrões de ISSR mostraram elevada
diversidade genética entre os recentes isolados Portugueses, sugerindo a
possibilidade de uma nova introdução. As árvores filogenéticas dos genes da
celulase e cytb sugeriram uma origem Asiática para os isolados Portugueses; ABSTRACT: Entomopathogenic nematodes are used as biocontrol agents. To understand
their diversity, a survey was undertaken in Portugal. Five species, namely
Steinernema feltiae, S. intermedium, S. kraussei, Steinernema sp. and
Heterorhabditis bacteriophora were identified. The ITS, 28S rRNA D2D3 region,
COXI and cytb sequences, used to study the genetic diversity of the two most
abundant species, S. feltiae and H. bacteriophora, showed no significant
differences among the isolates.
Bursaphelenchus xylophilus causes severe disease in pine trees and was
detected for the first time in Europe and in Portugal in 1999. To evaluate the
genetic diversity of Portuguese isolates and identify disease spread pathways,
the sequence of 5.8S rRNA IGS region, cytb and cellulase genes, combined
with ISSR fingerprints were used. ISSR fingerprints show a high genetic
variability among recent Portuguese isolates, suggesting the possibility of a new
introduction. Phylogenetic trees based on cellulase and cytb genes suggests an
Asian origin for Portuguese isolates
Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchidae) associated with Hylobius sp. from Pinus pinaster in Portugal
Bursaphelenchus antoniae sp. n. is described and illustrated. Dauer juveniles were isolated from the body of the large pine
weevil, Hylobius sp., collected from maritime pine (Pinus pinaster) stumps, in Portugal. Bursaphelenchus antoniae sp. n. was reared
and maintained in P. pinaster wood segments and on Petri dish cultures of the fungi Botrytis cinerea and Monilinia fructicola. The new
species is characterised by a relatively small body length of ca 583 μm (females) and 578 μm (males), a lateral field with two incisures,
presence of a small vulval flap and a conoid female tail with a rounded or pointed terminus. Males have stout spicules with a disc-like
cucullus and seven caudal papillae arranged as a single midventral precloacal papilla, one precloacal pair and two postcloacal pairs. In
the character of the lateral field, B. antoniae sp. n. comes close to B. abietinus, B. rainulfi and B. hylobianum, whilst spicule characters
place it within the piniperdae-group sensu Ryss et al. Morphologically, B. antoniae sp. n. is closest to B. hylobianum; the spicules of
these two species having flattened, wing-like, alae on the distal third of the lamina. Bursaphelenchus antoniae sp. n. is distinguished
from B. hylobianum on the arrangement of the caudal papillae (two vs three pairs). ITS-RFLP profiles and the failure to hybridise
support the separation of the two species. Phylogenetic analysis of the new species, based on the 18S rDNA sequence, supports the
inclusion of this new species in the B. hylobianum-group sensu Braasch. Sequence analysis of the 28S rDNA D2/D3 domain did not
place the new species in a definite group
Detailed morphobiometric studies of Bursaphelenchus xylophilus and characterisation of other Bursaphelenchus species (Nematoda: Parasitaphelenchidae) associated with Pinus pinaster in Portugal
Detailed studies on Bursaphelenchus xylophilus are provided in this contribution. Comparative observations
between field and cultured populations of this species demonstrated significant size differences: cultured specimens
overall displayed larger size in all morphometric parameters. A principal component analysis (PCA) of the individuals
undergoing moulting allowed their separation in four groups namely J2-J3, J3-J4, J4F-F, and J4M-M; gonad length mean values
of these four groups made possible to distinguish the non-moulting groups J2, J3, J4F, J4M and adults. Seven Bursaphelenchus
species (B. hellenicus, B. leoni, B. pinasteri, B. sexdentati, B. teratospicularis, B. tusciae and B. xylophilus), associated with Pinus
pinaster in Portugal, were charaterized, including biometrical measurements and ratios as well excised spicules observed
under SEM; furthermore, B. hellenicus, B. pinasteri, B. sexdentati, B. tusciae and B. xylophilus were characterised on the
basis of their ITS-RFLP profiles. B. sexdentati and B. xylophilus were the only species found in high numbers in some of
the samples
First report of an entomopathogenic nematode from continental Portugal
In continental Portugal no information is available concerning
the occurrence of entomopathogenic nematodes.
During a survey in several different habitats from the
southern regions of the country, several isolates where
identified as Steinernema feltiae. This represents the first
report of an entomopathogenic nematode genus for continental
Portugal
The pine wood nematode, Bursaphelenchus xylophilus, in Portugal: possible introductions and spread routes of a serious biological invasion revealed by molecular methods
The pine wood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), is a major
world-wide pathogen and pest of pine, with impacts on forest health, natural ecosystem stability and international trade. In Portugal,
PWN was first diagnosed in 1999, the first occurrence also for Europe. The disease was recently detected on the island ofMadeira and in
northern Spain. In an attempt to search for more reliable and robust molecular markers that enable the study of intraspecific variability
of B. xylophilus from different geographic locations, the intergenic spacer (IGS) region of the 5S rRNA gene and inter-simple sequence
repeats (ISSR) analysis were used to determine the genetic relationships among 43 B. xylophilus isolates from Portugal, China, Japan,
South Korea and USA. IGS sequence analysis showed that this region can only be used to establish interspecific relationships, since no
differences were detected among Portuguese isolates from different geographic locations. Fingerprints obtained with ISSR show high
genetic variability among Portuguese isolates, except for the ones obtained prior to 2008. The ISSR dendrogram suggests the spread
of the disease inside continental Portugal and to Madeira. Until 2008, B. xylophilus populations found in continental Portugal showed
low genetic diversity, pointing to a single introduction, probably from Asia, whereas recent populations from continental Portugal
(2009-2010) and Madeira show high genetic diversity, suggesting multiple introductions from different origins
Three species of entomopathogenic nematodes of the family Steinernematidae (Nematoda: Rhabditida) new to continental Portugal
Abstract
In order to determine the species of entomopathogenic nematodes in Continental Portugal, a survey was conducted in the country. Nematodes were recovered from soil samples collected from Alentejo, Algarve, center and the north of Portugal from 2006-2009. Isolates were identified based on morphology and sequence analysis. Phylogenetic analysis was based on sequences of partial 28S (D2D3), internal transcribed spacer (ITS) and cytochrome oxidase c subunit I gene (COX I). Isolates 59F, 15G, 20F and 2B were characterized in detail. Isolate 2B shows a morphology identical to Steinernema intermedium which is a member of the affine/intermedium-group and is characterized by the presence of strongly curved and robust spicules with a distinct rostrum, in the male, and dorsal tail depression in third-stage infective junveniles (IJ). However, the phylogenies based on the three molecular markers revealed that isolate 2B is more closely related to S. affine than with S. intermedium. The morphological results for isolate 20F identify it as S. kraussei which is a member of kraussei/feltiae–group, characterized by IJs with a straight body of medium length (mean = 700–950 μm), lateral field mostly with eight ridges, rather broad, flatly rounded and continuous cephalic region, excretory pore at level of mid-pharynx; males with mucronate tail, yellowish spicules ca. 50 μm long and wide manubria; females with short conoid tail with pointed non-mucronate tip. Based on morphology and sequence analysis, isolates 59F and 15G were considered conspecific and identified as a species belonging to the glaseri–group. More detailed studies are necessary to solve if these isolates represent a new species
Can functional hologenomics aid tackling current challenges in plant breeding?
Molecular plant breeding usually overlooks the genetic variability that arises from the association of plants with endophytic
microorganisms, when looking at agronomic interesting target traits. This source of variability can have crucial effects on
the functionality of the organism considered as a whole (the holobiont), and therefore can be selectable in breeding
programs. However, seeing the holobiont as a unit for selection and improvement in breeding programs requires novel
approaches for genotyping and phenotyping. These should not focus just at the plant level, but also include the associated
endophytes and their functional effects on the plant, to make effective desirable trait screenings. The present review in tends to draw attention to a new research field on functional hologenomics that if associated with adequate phenotyping
tools could greatly increase the efficiency of breeding programs.info:eu-repo/semantics/publishedVersio
First report of the genus Heterorhabditis (Nematoda: Heterorhabditidae) from continental Portugal
Until recently, the only entomopathogenic nematode (EPN) species reported from continental Portugal, was of the genus Steinernema. Following a national survey of EPNs in continental Portugal, several natural and managed habitats have been surveyed in the southern part of the country. From 57 soil samples collected using the Galleria mellonella trapping method, three samples yielded EPN. Morphological characterization and sequence analysis of the ITS regions of ribosomal DNA allowed the identification of EPN isolates as Heterorhabditis bacteriophora, representing the first report of this genus for continental Portugal
Screening of natural Wolbachia infection in mosquitoes (Diptera: Culicidae) from the Cape Verde islands
Funding Information: We are grateful to the National Institute of Public Health for the laboratory support in Cape Verde, and to technicians from the Ministry of Health for their assistance in field work. We would like to thank Prof. Paulo Almeida for providing DNA controls of Cx. pipiens and Cx. quinquefasciatus used in the species identification PCR assay. Funding Information: This work was funded by national funds through FCT—Fundação para a Ciência e Tecnologia, I.P., within the framework of the project ARBOMONITOR (PTDC/BIA-OUT/29477/2017. Aires da Moura was funded by the Ph.D. fellowship program of Camões I.P. Publisher Copyright: © 2023, The Author(s).Background: Wolbachia pipientis is an endosymbiont bacterium that induces cytoplasmic incompatibility and inhibits arboviral replication in mosquitoes. This study aimed to assess Wolbachia prevalence and genetic diversity in different mosquito species from Cape Verde. Methods: Mosquitoes were collected on six islands of Cape Verde and identified to species using morphological keys and PCR-based assays. Wolbachia was detected by amplifying a fragment of the surface protein gene (wsp). Multilocus sequence typing (MLST) was performed with five housekeeping genes (coxA, gatB, ftsZ, hcpA, and fbpA) and the wsp hypervariable region (HVR) for strain identification. Identification of wPip groups (wPip-I to wPip-V) was performed using PCR–restriction fragment length polymorphism (RFLP) assay on the ankyrin domain gene pk1. Results: Nine mosquito species were collected, including the major vectors Aedes aegypti, Anopheles arabiensis, Culex pipiens sensu stricto, and Culex quinquefasciatus. Wolbachia was only detected in Cx. pipiens s.s. (100% prevalence), Cx. quinquefasciatus (98.3%), Cx. pipiens/quinquefasciatus hybrids (100%), and Culex tigripes (100%). Based on the results of MLST and wsp hypervariable region typing, Wolbachia from the Cx. pipiens complex was assigned to sequence type 9, wPip clade, and supergroup B. PCR/RFLP analysis revealed three wPip groups in Cape Verde, namely wPip-II, wPip-III, and wPip-IV. wPip-IV was the most prevalent, while wPip-II and wPip-III were found only on Maio and Fogo islands. Wolbachia detected in Cx. tigripes belongs to supergroup B, with no attributed MLST profile, indicating a new strain of Wolbachia in this mosquito species. Conclusions: A high prevalence and diversity of Wolbachia was found in species from the Cx. pipiens complex. This diversity may be related to the mosquito's colonization history on the Cape Verde islands. To the best of our knowledge, this is the first study to detect Wolbachia in Cx. tigripes, which may provide an additional opportunity for biocontrol initiatives. publishersversionpublishe