Plum pox in north america: identification of aphid vectors and a potential role for fruit in virus spread

ABSTRACT Thirteen aphid species were tested for their ability to transmit Pennsylvania isolates of Plum pox virus (PPV) collected in Columbia (PENN-3), Franklin (PENN-4), and York (PENN-7) Counties, PA. Four species, Aphis fabae, A. spiraecola, Brachycaudus persicae, and Myzus persicae, consistently transmitted PPV in preliminary transmission tests. Two species, Metopolophium dirhodum and Rhopalosiphum padi, were occasional inefficient vectors. Toxoptera citricida, from Florida, also was an effective vector but it does not occur in major stone-fruit-growing states. Species not transmitting PPV in parallel tests included Acyrthosiphon pisum, Aphis glycines, Aulacorthum solani, Macrosiphum euphorbiae, Rhopalosiphum maidis, and Sitobion avenae. When given a 3-day probing access period simultaneously on PPV-infected peach seedlings and healthy peach seedlings, Myzus persicae, Aphis spiraecola, A. fabae, and B. persicae transmitted PPV to 63, 31, 38, and 32% of the healthy peach seedlings, respectively. When given a similar probing period on PPV-infected peach fruit and healthy peach seedlings, the same aphid species transmitted PPV to 50, 35, 0, and 0% of seedlings, respectively. Results support the hypothesis of secondary PPV spread by indigenous aphids in Pennsylvania, and suggest that PPV-infected fruit has the potential to function as a virus source for long-distance dispersal.     

Diversity of Plum pox virus isolates in Bosnia and Herzegovina

Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K₁ with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K₁ typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period.     

Serological characterization of Italian isolates of plum pox potyvirus1

An Italian isolate of plum pox potyvirus (PPV) from apricot, Ispave 17, was used as antigen for production of monoclonal antibodies. Six clones secreting specific antibodies to PPV were obtained. All these monoclonal antibodies were used to test a collection of different Italian PPV isolates, collected from plum, apricot and peach orchards, and other European isolates (including PPV-D and PPV-M serotypes), using DAS-ELISA, SDS-PAGE, western blot and GIEM. In western blot analysis, the PPV-M and PPV-D coat protein, detected directly from crude peach GF305 extracts, showed different electrophoretic mobility, the coat protein of PPV-M being slightly larger than that of PPV-D. ELISA tests, performed with fixed dilutions of antibodies and limiting dilutions of clarified samples, showed with some monoclonal antibodies a marked difference between PPV-M and PPV-D strains, at ratios greater than 1:40 (w/v). Also in GIEM some monoclonal antibodies gave a good labelling reaction only with PPV-D serotype. With the help of this differentiation, it was found that all Italian isolates tested were of the D serotype and none of the severe M strain of PPV, which has not been reported in Italy.     

A Natural Population of Recombinant Plum Pox Virus is Viable and Competitive under Field Conditions

The isolate BOR-3, collected in Slovakia in 1996, was recently identified as a natural recombinant between an M and D type of Plum pox virus (PPV). Biological assays demonstrated its capacity to be aphid- and graft-transmitted to various Prunus spp. hosts. A study was carried out to determine the further presence of PPV recombinants in two epidemiologically distinct areas – Slovakia and France. Tools based on PPV-M and D subgroup typing, targeting P3–6K₁, CI and CP regions of the PPV genome were used for recombinant identification. Closely related recombinant variants were detected in different Prunus spp. during a survey conducted in Slovakia in 2001, but not within a set of selected PPV isolates from France collected between 1985 and 2001. Sequence analysis of the (Cter)NIb–(Nter)CP region of 10 recombinant isolates from Slovakia showed their high homology, reaching more than 98%. All the recombinant isolates shared the same recombination breakpoint situated in the C terminus of the NIb gene. Our study demonstrates that the PPV recombinants are viable and competitive with conventional PPV-M and D isolates. The present work indicates that the occurrence of recombinants within PPV isolates might be more common than previously assumed.     

Laboratory evaluation of temperature effects on the germination and growth of entomopathogenic fungi and on their pathogenicity to two aphid species

As part of an approach to select potential mycoinsecticides for aphid biocontrol, we investigated the effects of temperature on the growth, germination and pathogenicity of some hyphomycete fungi. Commercially available mycoinsecticides (based on Beauveria bassiana (Balsamo) Vuillemin and Verticillium lecanii (Zimmermann) Viegas) and other isolates of B bassiana, V lecanii, Metarhizium anisopliae (Metschnikoff) Sorokin and Paecilomyces fumosoroseus (Wize) Brown & Smith were evaluated. The rate of in vitro conidial germination of all isolates was slower at 10 and 15 degrees C than at 20 and 25 degrees C. Similarly, in vitro growth of most isolates was adversely affected at 10 and 15 degrees C. The greatest reduction at 10 degrees C in rates of conidial germination and colony growth, compared with other temperatures, was for M anisopliae isolates. Germination of V lecanii (isolate HRI 1.72) was fastest at 10 degrees C compared with the other fungi. It was also the most pathogenic of three isolates tested against Aphis fabae Scopoli and Myzus persicae Sulzer at 10, 18 and 23 degrees C. Generally, A fabae was more susceptible than M persicae to infection by the fungal isolates tested. A significant interaction between aphid species and temperature indicated that the pathogenic nature of an isolate was dependent not only on the target aphid species but also the temperature conditions of the bioassay. The series of studies, detailed above, allowed a temperature profile to be formed for the different isolates. Verticillium lecanii isolate HRI 1.72 (commercialised as Vertalec) was the most promising isolate selected from results of the series of experiments. Temperature profiles in conjunction with infectivity assays can be useful in selecting appropriate isolates for a particular thermal environment.     

Stability of the aphid transmission phenotype in cucumber mosaic virus

The stability of the aphid transmission phenotype in seven field isolates of cucumber mosaic virus (CMV) was studied, using aphids Aphis gossypii and Myzus persicae . Field isolates, obtained from four vegetable crops, were propagated in squash and Nicotiana glutinosa , and passaged by either aphid transmission or mechanical transfer. All seven isolates were transmissible by both aphids and this aphid transmission phenotype was stable after 20–24 mechanical passages. Upon further mechanical passaging, one of the seven isolates, CMV-2 A1-MT 60x, lost its transmissibility by Myzus persicae but was still transmissible by Aphis gossypii , although at a reduced rate. Isolates maintained by both aphid transmission and mechanical transfer were transmitted more efficiently by Aphis gossypii than by Myzus persicae . A comparison of the RNA profiles showed no major differences among the CMV isolates before and after mechanical passage.     

侵染垂花悬铃花的木尔坦棉花曲叶病毒分子特征研究

 垂花悬铃花曲叶病是近期在广东发现的一种新病害,病株表现为叶片向上卷曲,叶脉肿大,叶脉变深绿色等症状。PCR检测结果显示, 该病样中均存在菜豆金色花叶病毒属病毒。基因克隆及序列分析结果表明,该病毒分离物（GD11）DNA-A全长为2 737 nt,具有菜豆金色花叶病毒属病毒基因组典型特征,为闭合环状单链DNA,编码6个ORFs;该序列与木尔坦棉花曲叶病毒(CLCuMV)各分离物序列的相似性均大于89.0%,其中与G6、Okra06及GX1等分离物序列的相似性大于99.0%。该病毒分离物也伴有卫星DNA β分子,其全长为1 348 nt,与CLCuMV各分离物的DNA β序列相似性大于85.0%,其中与G6、Okra06及GX1等DNA β的序列相似性均大于99.0%。因此,侵染广东垂花悬铃花的病毒分离物属于CLCuMV,且与入侵我国的朱槿分离物G6、黄秋葵分离物Okra06及棉花分离物GX1亲缘关系很近。本文首次报道了CLCuMV及其卫星β复合侵染垂花悬铃花。     

Cross reactions of an antiserum to plum pox potyvirus1

In the early spring of 1992, plum pox-like viruses (PPLVs) were detected by standard ELISA in some Prunus species. The isolates reacted positively with plum pox potyvirus (PPV) antisera in immunosorbent electron microscopy and Western blot analysis. In Western blot analyses, bands associated with the coat protein subunits of the PPLVs were 48–56 kDa, whereas bands associated with the coat protein subunits of known PPV isolates were 32–37 kDa in size. Also, the PPLVs differed from known PPV isolates in their symptoms on woody and herbaceous indicators, and in their herbaceous host range. None of these PPLVs appears to be an isolate of PPV.     

An unusual isolate of turnip mosaic potyvirus fromAbutilon theophrasti in Piedmont, Italy

An isolate of the poty virus turnip mosaic virus (TuMV-Ab) showing severe mosaic symptoms inAbutilon theophrasti from Piedmont (northwestern Italy) in 1993, has been found to be of an unusual pathotype and serotype. The isolate was easily transmitted byAphyis gossiypii and Myzus persicae and was not seed-transmitted inA. Theophrasti. The host range of TuMV-Ab was different from that of another Piedmont isolate of TuMV fromAlliaria officinalis and from a TuMV isolate fromBrassica napus. TuMV-Ab was characterized using the reactions on the fourB. Napus lines S4, R4, 165 and S1 as the rare pathotype 7, found only once previously in Europe. Tests with polyclonal antisera indicated that TuMV-Ab was only distantly related to the two other TuMV isolates. Serological characterization with a panel of 30 monoclonal antibodies showed that TuMV-Ab belonged to one of the less common serotypes (JPN).     

BBTV两个株系DNA组分1的克隆及序列分析

 对在生物学特性特别是寄主范围上存在明显差异的NSP株系和NS株系的代表分离物(广州天河分离物和高州分离物)的DNA组分1进行了克隆和序列分析,结果表明:2个代表分离物的DNA组分1全序列、ORF及其编码的氨基酸序列的变异率分别为3.2%、3.1%和2.8%,从而进一步确证了可以用寄主范围来作上述2个株系的鉴定。此外,这2个株系的DNA组分1、ORF及其编码的氨基酸序列分别与肖火根等报道的中国(广东)分离物、以及亚洲组和南太平洋组各分离物进行比较,结果表明:NS株系与肖火根等报道的中国(广东)分离物的亲缘关系很密切;这2个株系与亚洲组各分离物的差异均较小,而与南太平洋组各分离物的差异均较大,它们应属亚洲组。     

Transmission efficiency of Papaya ringspot virus by three aphid species

The transmission efficiency of Papaya ringspot virus (PRSV) by three aphid vectors (i.e., Aphis gossypii, A. craccivora, and Myzus persicae) was studied. Efficiency was measured by single-aphid inoculation, group inoculation (using five aphids), duration of virus retention, and the number of plants following a single acquisition access period (AAP) to which the aphids could successfully transmit the virus. Single-aphid inoculation studies indicated that M. persicae (56%) and A. gossypii (53%) were significantly more efficient in transmitting PRSV than A. craccivora (38%). Further, in the former two species, the time required for initiation of the first probe on the inoculation test plant was significantly shorter compared to A. craccivora. PRSV transmission efficiency was 100% in all three species when a group of five aphids were used per plant. There was a perceptible decline in transmission efficiency as the sequestration period increased, although M. persicae successfully transmitted PRSV after 30 min of sequestration. A simple leaf-disk assay technique was employed for evaluating the transmission efficiency of three species of aphids. The results of leaf-disk assays also indicated that A. gossypii (48%) and M. persicae (56%) were more efficient PRSV vectors than A. craccivora. Using leaf-disk assays, the ability of individual aphids to inoculate PRSV serially to a number of plants was studied. Following a single AAP on an infected leaf, M. persicae was more efficient than the other two species with 52.5% transmission after the first inoculation access period (IAP). However, its inoculation efficiency significantly decreased with the second and subsequent IAPs. A. gossypii was able to transmit PRSV sequentially up to four successive leaf disks, but with significantly declining efficiency. Since A. gossypii is reported to be the numerically dominant vector in south India in addition to being a more efficient vector capable of inoculating PRSV to multiple plants, it should be the target vector for control strategies.     

北京地区侵染茄子的番茄斑萎病毒分子鉴定与分析

调查发现北京地区一温室栽培茄子Solanum melongena L.出现严重病毒病。利用基于小RNA的高通量测序技术和RT-PCR方法,明确了引起茄子病害的病毒种类为番茄斑萎病毒,将其命名为TSWV-eggplant分离物。进一步克隆了该病毒的基因组全长(S RNA、M RNA、L RNA),并构建其系统发育树。结果表明,该分离物的S RNA与美国分离物亲缘关系较近,M RNA与中国分离物亲缘关系较近,而L RNA与韩国分离物亲缘关系较近。因此,本研究发现的TSWV分离物与国内已发生报道的分离物不同,该分离物是否存在不同分离物之间基因组的重组需要进一步研究。     

Genetic and Morphological Diversity of Temperate and Tropical Isolates of Phytophthora capsici

ABSTRACT Phytophthora capsici is a diverse species causing disease on a broad range of both temperate and tropical plants. In this study, we used cultural characteristics, amplified fragment length polymorphism (AFLP), and DNA sequence analyses of the ribosomal internal transcribed spacer (ITS) region and mitochondrial cytochrome oxidase II (cox II) genes to characterize temperate and tropical isolates from a wide range of host species. All but one temperate isolate grew at 35 degrees C, while all tropical isolates did not. All but two tropical isolates formed chlamydospores, while temperate isolates did not. There was strong bootstrap support for separation of temperate and tropical isolates using AFLP analysis; however, the temperate isolates appeared as a subgroup within the observed variation of the tropical isolates. The majority of temperate isolates clustered within a single clade with low variation regardless of host or geographical origin, while the tropical isolates were more variable and grouped into three distinct clades. Two clades of tropical isolates grouped together and were affiliated closely with the temperate isolates, while the third tropical clade was more distantly related. Phylogenetic analysis of the ITS regions resulted in similar groupings and variation within and between the temperate and tropical isolates as with the AFLP results. Sequence divergence among isolates and clades was low, with more variation within the tropical isolates than within the temperate isolates. Analysis of other species revealed shorter branch lengths separating temperate and tropical isolates than were observed in comparisons among other phylogenetically closely related species in the genus. Analysis of cox II sequence data was less clear. Although the temperate and tropical isolates grouped together apart from other species, there was no bootstrap support for separating these isolates. Restriction fragment length polymorphism (RFLP) analysis of the ITS regions separated the temperate and tropical isolates, as in the AFLP and ITS phylogenetic analyses. However, RFLP analysis of the cox I and II gene cluster did not distinguish between temperate and tropical isolates. The differences in grouping of isolates in these two RFLP studies should be helpful in identifying isolate subgroups. Our data do not fully clarify whether or not temperate and tropical isolates should be separated into different species. The available worldwide data are incomplete and the full range of variation in the species is not yet known. We suggest refraining from using the epithet P. tropicalis until more data are available.     

Host Specialization in the Charcoal Rot Fungus, Macrophomina phaseolina

ABSTRACT To investigate host specialization in Macrophomina phaseolina, the fungus was isolated from soybean, corn, sorghum, and cotton root tissue and soil from fields cropped continuously to these species for 15 years in St. Joseph, LA. Chlorate phenotype of each isolate was determined after growing on a minimal medium containing 120 mM potassium chlorate. Consistent differences in chlorate sensitivity were detected among isolates from different hosts and from soil versus root. To further explore genetic differentiation among fungal isolates from each host, these isolates were examined by restriction fragment length polymorphism and random amplified polymorphic DNA (RAPD) analysis. No variations were observed among isolates in restriction patterns of DNA fragments amplified by polymerase chain reaction covering the internal transcribed spacer region, 5.8S rRNA and part of 25S rRNA, suggesting that M. phaseolina constitutes a single species. Ten random primers were used to amplify the total DNA of 45 isolates, and banding patterns resulting from RAPD analysis were compared with the neighbor-joining method. Isolates from a given host were genetically similar to each other but distinctly different from those from other hosts. Chlorate-sensitive isolates were distinct from chlorate-resistant isolates within a given host. In greenhouse tests, soybean, sorghum, corn, and cotton were grown separately in soil infested with individual isolates of M. phaseolina that were chosen based on their host of origin and chlorate phenotype. Root colonization and plant weight were measured after harvesting. More colonization of corn roots occurred when corn was grown in soil containing corn isolates compared with isolates from other hosts. However, there was no host specialization in isolates from soybean, sorghum, or cotton. More root colonization in soybean occurred with chlorate-sensitive than with chlorate-resistant isolates.     

Causal agent of sharka disease: new and emerging events associated with Plum pox virus characterization

Plant RNA viruses have a high genetic variation potential due to the absence of proofreading activity in their RNA replicase. In addition to mutation, recombination is generally thought to be an important source of variability. Both evolutionary processes have contributed to the diversity of Plum pox virus (PPV). There are now six recognized subgroups, strains or serotypes of PPV (D, M, Rec, EA, C and W). Isolates belonging to the PPV-Rec subgroup are derived from RNA recombination between PPV-D and PPV-M and occur frequently in various central and eastern European countries. The divergent isolate W3174 is a new and distinct strain of PPV, identified as PPV-W. It is quite conceivable that, with time, other groups will be defined and that the present classification will need revision to accommodate additional PPV variability.     

Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

Genetic diversity and molecular epidemiology of the T strain of Plum pox virus

Very limited information is available on the origin, diversity and evolution of Plum pox virus (PPV) ‘Turkey’ (T) strain. Phylogenetic analyses based on partial sequences of 421 isolates and complete genome sequences of 57 isolates, representing the geographical distribution of PPV-T in Turkey, revealed the existence of several monophyletic and, in some cases, geographically limited groups within the PPV-T strain (Ankara-Konya1-Kayseri, Ankara-Balkan, Istanbul, Konya2 and Balkan). PPV-T diversity (0.018%) was found to be greater than that of PPV strains D and Rec but lower than that of the M strain when including the newly described and divergent M-Istanbul isolates, suggesting a long evolutionary history for PPV-T. The European part of Turkey in the Balkans, close to Bulgaria where PPV was identified for the first time, appears as a likely centre of origin for PPV-T isolates. The colonization of various parts of Turkey by diverse isolates from that region, followed by secondary local spread, is the most likely scenario for the diffusion of PPV-T in Turkey.