Biological, serological and molecular characterization of three isolates of Citrus tristeza closterovirus introduced into Morocco

The biological, serological and genomic diversity of three Citrus tristeza virus (CTV) isolates from various geographical regions was studied: isolate P1 from lemon cv. 'Meyer' in a field near Marrakech (MA) in 1983, and isolates P2 and R1 detected in imported Spanish clementine germplasm by the Moroccan NPPO in 1998 and 2000. P1 induced severe vein clearing on Mexican lime and grapefruit, mild stem pitting on Mexican lime and moderate stem pitting on grapefruit. P2 and R1 only induced mild vein clearing on Mexican lime and caused no stem pitting or other symptoms on indicator plants used as controls. Only isolate P1 reacted with monoclonal antibody MCA-13, whereas all isolates reacted positively with the 3DF1+3CA5 mixture. The Moroccan clones P1–3 and P1–5, and all other severe isolates obtained from GenBank, showed a phenylalanine at amino acid position 124 of their coat protein sequences. This epitope confers MCA13 reactivity. The Spanish clones had tyrosine instead at this position. The deduced amino-acid sequence of coat protein of P1 clones clusters close to severe strains CB3–104 and FL7, respectively from Brazil and USA (Florida) (Group 5), whereas the sequences from P2 and R1 cluster close to typical strains from Portugal 25–120 and USA (Florida) T30 (Group M). The three techniques for distinguishing CTV isolates were clearly correlated.     

Biological, serological, and molecular variabilities of clover yellow vein virus

ABSTRACT A comparative study was made on the host reactions, serological properties, and nucleotide sequences of the coat protein (CP) gene of 10 clover yellow vein virus (C1YVV) isolates and one bean yellow mosaic virus (BYMV) isolate collected from different host plant species and locations in Japan. Two strains of C1YVV isolates, grouped on the basis of host reactions on Chenopodium amaranticolor, C. quinoa, Nicotianaclevelandii, N. benthamiana, Vicia faba, and Trifolium repens, corresponded to two serotypes determined by double-antibody sandwich- and triple-antibody sandwich-enzyme-linked immunosorbent assay using three polyclonal and nine monoclonal antibodies. These results were also confirmed by nucleotide sequence analysis of the CP gene. The CP gene of C1YVV isolates of strain 1, including the Australian isolate C1YVV-B, had 93 to 98% nucleotide identities and 97 to 99.6% amino acid identities. The CP of C1YVV isolates of strain 2, including the New Zealand isolate C1YVV-NZ, had 92 to 98% nucleotide identities and 95 to 98% amino acid identities. The nucleotide identities and the amino acid identities between the two C1YVV strains were 82 to 84%, and 90 to 94%, respectively. When compared with the CP sequences of 12 C1YVV isolates, the CP sequence of the BYMV isolate had 71 to 73% nucleotide identity and 73 to 77% amino acid identity. Amino acid sequence differences among C1YVV isolates from strains 1 and 2 were located mostly at the N-terminal regions of the CP. Our results indicated that the C1YVV isolates studied could be separated into two strains on the basis of host reactions, serology, and the nucleotide sequence of the CP gene.     

Biological, serological, and molecular differences among isolates of potato a potyvirus

ABSTRACT Sequences of the coat protein (CP) and 3'-end nontranslated region (3'NTR) of 13 isolates and the helper component proteinase (HC) of nine isolates of potato A potyvirus (PVA) were determined and compared with the eight previously determined PVA CP and 3'NTR sequences and one HC sequence. CP amino acid (aa), 3'NTR nucleotide, and HC aa sequence identities were 92.9, 93.4, and 94.8%, respectively. Sequence data, serological tests, and the necrotic local lesions induced in the leaves of the potato hybrid 'A6' confirmed that tamarillo mosaic virus is a strain of PVA. The aa substitutions A6T and G7S in the CP N-terminus were correlated with loss of aphid transmissibility. Development of necrotic lesions or nonnecrotic symptoms in the systemically infected leaves or lack of systemic spread in potato cv. King Edward were used to place the PVA isolates into four strain groups, but this grouping was not correlated with any differences in CP, HC, or 3'NTR. Recognition of CP by three monoclonal antibodies was used to place the PVA isolates into three groups different from the four groups above. The epitopes of two mono-clonal antibodies were mapped by site-directed mutagenesis to the same lysine residue at the CP aa 34.     

Biological, serological and molecular characterization of a cucumber mosaic virus isolate from India

A. virus causing mosaic and leaf deformation of Physalis minima has been identified as an isolate of cucumber mosaic virus (CMV) on the basis of its transmission by aphids in a non-persistent manner, polyhedral particles of 29 nm diameter, molecular weight of coat protein subunits us 24-5 kDa. serological relationship with a CMV isolate and a tripartite single-stranded RNA genome with a subgenomic RNA4- Furthermore. cDNA representing coat protein gene was synthesized and cloned. Complete nucleotide sequences (890 nt) were obtained which showed a coat protein gene open reading frame of 657 residues. THE nucleotide sequences provided the 218 amino ACID sequences of the coat protein. Nucleotide as well as amino acid sequences revealed more than 90% identity with the CMV subgroup I strains.     

Molecular and serological characterization of iris yellow spot virus, a new and distinct tospovirus species

ABSTRACT A new tospovirus was identified in iris cultivations in the Netherlands. Both serological comparisons and sequence determination of the S RNA demonstrate that this virus represents a new and distinct species, belonging to a separate serogroup, and for which the name iris yellow spot virus (IYSV) is proposed. The disease symptoms on iris are characterized by yellow spots on the leaves. Its experimental host range is very narrow and, in addition to iris, only includes Nicotiana benthamiana and Datura stramonium. The nucleoprotein of IYSV shows only 30 to 44% sequence identity with those of other tospoviruses identified so far; the highest homology being found with the tospovirus species of serogroup IV.     

Phylogenetic relationships and virulence assays of Fusarium secorum from sugar beet suggest a new look at species designations

Fusarium spp. are responsible for significant yield losses in sugar beet (Beta vulgaris) with Fusarium oxysporum f. sp. betae most often reported as the primary causal agent. Recently, a new species, F. secorum, was reported to cause disease in sugar beet but little is known on the range of virulence within F. secorum or how this compares to the virulence and phylogenetic relationships previously reported for Fusarium pathogens of sugar beet. To initiate this study, partial translation elongation factor 1-α (TEF1) sequences from seven isolates of F. secorum were obtained and the data were added to a previously published phylogenetic tree that includes F. oxysporum f. sp. betae. Unexpectedly, the F. secorum strains nested into a distinct group that included isolates previously reported as F. oxysporum f. sp. betae. These results prompted an expanded phylogenetic analysis of TEF1 sequences from genomes of publicly available Fusarium spp., resulting in the additional discovery that some isolates previously reported as F. oxysporum f. sp. betae are F. commune, a species that is not known to be a sugar beet pathogen. Inoculation of sugar beet with differing genetic backgrounds demonstrated that all Fusarium strains have a significant range in virulence depending on cultivar. Taken together, the data suggest that F. secorum is more widespread than previously thought. Consequently, future screening for disease resistance should rely on isolates representing the full diversity of the Fusarium population that impacts sugar beet.     

Generation of polyclonal antibodies and serological analyses of nucleocapsid protein of Soybean vein necrosis-associated virus: A distinct soybean infecting tospovirus serotype

Soybean vein necrosis-associated virus (SVNaV) is a newly isolated tospovirus from field-grown soybeans in the United States. Polyclonal antibodies generated against the recombinant Escherichia coli-expressed nucleocapsid protein (NP) of the virus, reacted specifically with SVNaV and exhibited low, if any, cross-reactivity with other species within the genus Tospovirus. The serological results are in agreement with low sequence homology of the SVNaV-NP gene when compared with those of other tospovirus species, some of which are capable of infecting soybean naturally. Phylogenetic analysis utilizing NP sequences from several SVNaV isolates collected from different geographical regions and various soybean genotypes over 4?years showed close relationships, but distinct from the representatives of all the established serogroups or distinct serotypes within the genus of Tospovirus. All SVNaV isolates examined reacted strongly with the generated polyclonal antibodies. Collectively, our serological analyses suggest that SVNaV represents a new and distinct soybean-infecting tospovirus serotype. Furthermore, our data demonstrate that antiserum against NP has the potential to serve as a reliable probe for SVNaV detection in field-grown soybeans.     

Molecular,biological and serological variability of Zucchini yellow mosaic virus in Tunisia

A study was conducted to better understand the population structure of Zucchini yellow mosaic virus (ZYMV), a severe virus affecting cucurbit crops worldwide, in Tunisia and to estimate whether the use of resistant cultivars may provide durable control. Analysis of the polymerase and coat protein (NIb‐CP) partial sequences of 83 isolates collected in the three main cucurbit‐growing areas in Tunisia showed that ZYMV grouped into two distinct clusters within ZYMV molecular group A. An important variability was observed in the MREK motif of the P3 protein, a motif associated with tolerance breaking in ZYMV‐tolerant zucchini squash cultivars. Interestingly, significant differences were found in the distribution of the MREK variants in the two clusters defined by the partial NIb‐CP sequences, MREK and MKEK sequences being more common in cluster 1 and cluster 2, respectively. When combining NIb‐CP and P3 sequence information, ZYMV molecular variability was shown to be significantly higher in the Cap Bon region than in the Bizerte area. An important biological variability was observed in a subset of 23 isolates regarding symptomatology in susceptible or resistant cucurbits. Some isolates overcame ZYMV tolerance or resistance in zucchini squash and melon, but not in cucumber. Three serotypes were differentiated using a set of 13 monoclonal antibodies (MAbs). Seven parameters characterizing the 23 isolates, including molecular, serological and biological properties, were used for a multiple component analysis (MCA). This analysis revealed that symptom intensity of a given isolate was similar in different susceptible cucurbit hosts, suggesting similar degrees of aggressiveness in different hosts.     

Biological and molecular characterization of Potato yellow blotch virus,a new species of the genus Potyvirus

A new species of the genus Potyvirus infecting potatoes, with the proposed name Potato yellow blotch virus (PYBV), was discovered in a breeding line 99m-022-026 in Scotland. The infected plants show isolated yellow blotches on the leaves. The genome of PYBV contains a large open reading frame encoding a single polyprotein of 3054 amino acids. Sequence analysis shows that PYBV is closely related to Potato virus A (PVA), with an overall 72% identity at the nucleotide level for the whole genome. The least conserved P1 protease gene shares only 50% nucleotide identity with PVA. The host range of PYBV was comparable to PVA on solanaceaous and non-solanaceous indicator plant species with the exception of Solanum demissum A and Y. Different symptoms were also observed for PYBV and PVA in Nicotiana benthamiana, Nicotiana hesperis and Nicotiana occidentalis P1. The susceptibility of potato (Solanum tuberosum) cultivars to PYBV and PVA was similar. In over 5 years of investigation, PYBV has not been found in commercial seed and ware potato crops in Scotland.     

Species diversity,phylogeny and genetic variability of begomovirus populations infecting leguminous weeds in northeastern Brazil

A survey of begomoviruses infecting leguminous weeds (family Fabaceae) was carried out in four states of northeastern Brazil. A total of 26 full‐length begomovirus components (19 DNA‐A and seven DNA‐B, with three pairs of cognate A and B components) were amplified using rolling‐circle amplification, then cloned and sequenced. Sequence analysis indicated the presence of six species, four of them novel. In phylogenetic analysis five of the viruses clustered with other Brazilian begomoviruses, but one of them (Euphorbia yellow mosaic virus, EuYMV) clustered with viruses from other countries in Central and South America. Evidence of recombination was found among isolates of Macroptilium yellow spot virus (MaYSV). The MaYSV population had a high degree of genetic variability. Macroptilium lathyroides was revealed as a common host for several of these viruses, and could act as a mixing vessel from which recombinant viruses could emerge. The results indicate that leguminous weeds are reservoirs of several begomoviruses in Brazil, and could play a significant role in begomovirus epidemics, both as inoculum sources and as sources of emerging novel viruses.     

Biological and molecular characterization of a distinct Citrus tristeza virus isolate originating from a lemon tree in Greece

A Citrus tristeza virus (CTV) isolate (L192GR) naturally occurring in lemon trees of more than 100 years old in Greece was fully characterized. Virus‐derived small interfering RNAs, induced by Dicer processing of dsRNAs formed during RNA virus replication, were isolated and used as targets for sequencing. Next‐generation high‐throughput sequencing using the Ion Torrent platform was performed. A total of 432 632 sequences, 94·05% of which corresponded to L192GR, were determined. Subsequent bioinformatics analysis enabled the determination of the full‐length 19 251 nt genome of the L192GR isolate (GenBank no. KC262793 ). Comparative analysis of complete genomes revealed molecular homology with CTV‐VT isolate FS2‐2 from Florida (GenBank no. EU937519 ) with 98·2% nucleotide sequence identity. Recombination events were detected in L192GR and they probably contribute to its unique characteristics. Specifically, although most isolates of the CTV‐VT group induce the seedling yellows syndrome and react positively with the monoclonal antibody MCA13, which is typically associated with severe CTV isolates, the MCA13‐positive L192GR gave very mild or even no symptoms in the seedling yellows indicator plants. Furthermore, experimental aphid transmissibility studies revealed a poor transmission efficiency of 20%. This is the first report of a CTV isolate originating from a lemon tree being fully characterized at biological, serological and molecular levels. The present study further confirms that, when the goal is the risk assessment associated with a new pathogen or isolate in a particular area, molecular data have to be combined with the biological properties of the pathogen.     

三种重要皮蠹科害虫的幼虫形态与分子鉴定

为寻求白腹皮蠹和赤毛皮蠹幼虫简便科学的鉴定方法,利用形态学结合分子生物学对其做了鉴定研究,首先对其触角、上内唇、臀突和胸足胫节的特征进行了形态描述;再分别提取白腹皮蠹、赤毛皮蠹和外群黑毛皮蠹幼虫的虫体DNA进行PCR扩增,通过测序比对构建了系统进化树。结果表明:白腹皮蠹幼虫上内唇的中间2根缘刚毛呈蘑菇状,粗短,而赤毛皮蠹幼虫的则呈矛状,细长,二者上内唇的形态特征差异非常明显,可用于二者形态上的鉴定;对3种皮蠹幼虫的序列进行比对,白腹皮蠹与赤毛皮蠹的碱基相似率为89.97%,黑毛皮蠹与白腹皮蠹和赤毛皮蠹的碱基相似率分别为77.54%和77.05%,差异均明显,可用于3种皮蠹幼虫种间鉴定的线粒体COⅠ基因标记序列。     

Oidium neolycopersici: intraspecific variability inferred from amplified fragment length polymorphism analysis and relationship with closely related powdery mildew fungi infecting various plant species

Previous works indicated a considerable variation in the pathogenicity, virulence, and host range of Oidium neolycopersici isolates causing tomato powdery mildew epidemics in many parts of the world. In this study, rDNA internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) patterns were analyzed in 17 O. neolycopersici samples collected in Europe, North America, and Japan, including those which overcame some of the tomato major resistance genes. The ITS sequences were identical in all 10 samples tested and were also identical to ITS sequences of eight previously studied O. neolycopersici specimens. The AFLP analysis revealed a high genetic diversity in O. neolycopersici and indicated that all 17 samples represented different genotypes. This might suggest the existence of either a yet unrevealed sexual reproduction or other genetic mechanisms that maintain a high genetic variability in O. neolycopersici. No clear correlation was found between the virulence and the AFLP patterns of the O. neolycopersici isolates studied. The relationship between O. neolycopersici and powdery mildew anamorphs infecting Aquilegia vulgaris, Chelidonium majus, Passiflora caerulea, and Sedum alboroseum was also investigated. These anamorphs are morphologically indistinguishable from and phylogenetically closely related to O. neolycopersici. The cross-inoculation tests and the analyses of ITS sequences and AFLP patterns jointly indicated that the powdery mildew anamorphs collected from the above mentioned plant species all represent distinct, but closely related species according to the phylogenetic species recognition. All these species were pathogenic only to their original host plant species, except O. neolycopersici which infected S. alboroseum, tobacco, petunia, and Arabidopsis thaliana, in addition to tomato, in cross-inoculation tests. This is the first genome-wide study that investigates the relationships among powdery mildews that are closely related based on ITS sequences and morphology. The results indicate that morphologically indistinguishable powdery mildews that differed in only one to five single nucleotide positions in their ITS region are to be considered as different taxa with distinct host ranges.     

Biotic, molecular, and phylogenetic characterization of bean calico mosaic virus, a distinct begomovirus species with affiliation in the squash leaf curl virus cluster

ABSTRACT Bean calico mosaic virus (BCMoV), a whitefly-transmitted geminivirus from Sonora, Mexico, was purified, and the genome components were cloned and sequenced. Purified viral fractions and cloned genome components were infectious by biolistic inoculation to bean, completing Koch's postulates for both. The B biotype of the whitefly Bemisia tabaci efficiently transmitted both native virus and progeny virus derived from cloned DNA inoculum. Host ranges of native virus and of progeny virus derived from cloned DNA were identical based upon whitefly and biolistic mediated transmission, respectively. BCMoV has a relatively wide experimental host range among begomoviruses known to infect bean, encompassing genera and species within the Fabaceae, Malvaceae, and Solanaceae. BCMoV has a bipartite genome, as do other New World begomoviruses. BCMoV DNA-A shared highest nucleotide sequence identities with squash leaf curl virus-E strain (SLCV-E) and cabbage leaf curl virus (CaLCV) at 80.1 and 80.7%, respectively. BCMoV DNA-B shared highest nucleotide sequence identity with SLCV-E at 70.7%. The common region (CR) sequences of BCMoV and SLCV-E are 73 to 76% identical; however, modular cis-acting elements within the CR involved in replication origin function and recognition are 100% conserved. Phy-logenetic analysis indicated that BCMoV DNA-A shares a most recent common ancestor with the DNA-A of two viruses that also occur in the Sonoran Desert, SLCV-E and Texas pepper virus (TPV-TAM), and CaLCV from Florida. In contrast, a phylogenetic analysis indicated that BCMoV DNA-B shares a most recent common ancestor with SLCV-E; whereas DNA-B of CaLCV clustered in a separate clade with pepper hausteco virus. Collectively, biological and molecular characteristics indicate that BCMoV is a distinct begomovirus species with the northernmost distribution of any begomovirus isolated from bean in the Americas. Furthermore, the phylogenetic relationships of begomovirus cognate components are not necessarily identical, suggesting that DNA-A and DNA-B of some begomoviruses may have different evolutionary histories.     

Pathogenicity, symptom development, and mycotoxin formation in wheat by Fusarium species frequently isolated from sugar beet

Crop rotations with putative non-host crops such as sugar beet are often recommended to reduce Fusarium head blight (FHB) in cereals. However, recent observations have shown pathogenic, endophytic, and saprotrophic colonization of sugar beet with various Fusarium spp. Therefore, strains of seven species frequently isolated from sugar beet were tested for pathogenicity on wheat. Species-specific symptoms on heads and kernels were evaluated and the grains were analyzed for 20 mycotoxins with liquid chromatography-tandem mass spectrometry. Fusarium graminearum, F. culmorum, and F. cerealis from sugar beet caused typical FHB symptoms and mycotoxin contamination with deoxynivalenol and nivalenol, while a high incidence of black point was observed in heads inoculated with F. tricinctum or F. equiseti. Black point kernels revealed 3.4 to 14.5 times higher mycotoxin concentrations than symptomless grains, containing enniatin B1 at 38,000 μg/kg, moniliformin at 4,900 μg/kg, and 2-amino-14,16-dimethyloctadecan-3-ol at 5,500 μg/kg, as well as monoacetoxyscirpenol at 2,600 μg/kg and nivalenol at 3,800 μg/kg. Monitoring of these latter two species in the field is hampered by the lack of typical head symptoms after infection. In further experiments, the impact of sugar beet residues on FHB severity and the correlation between mycotoxin contamination of cereal lots and the amount of black point have to be evaluated.     

Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.