Two paths of sequence divergence in the citrus tristeza virus complex

ABSTRACT Comparison of a sampling of complementary DNA (cDNA) sequences from the Florida citrus tristeza virus (CTV) isolates T3 and T30 to the sequence of the genome of the Israeli isolate VT showed a relatively consistent or symmetrical distribution of nucleotide sequence identity in both the 5' and 3' regions of the 19.2-kb genome. In contrast, comparison of these sequences to the sequence of isolate T36 showed a dramatic decrease in sequence identity in the 5' proximal 11 kb of the genome. A cDNA probe derived from this region of the T36 genome hybridized to double-stranded RNA (dsRNA) of only 3 of 10 different Florida CTV isolates. In contrast, analogous probes from T3 and T30 hybridized differentially to the seven isolates not selected by the T36 probe. Primers designed from cDNA sequence for polymerase chain reaction (PCR) selectively amplified these 10 isolates, allowing them to be classified as similar to T3, T30, or T36. In contrast, individual cDNA probes derived from the 3' terminal open reading frames of the T3, T30, and T36 genomes all hybridized to dsRNA from all Florida CTV isolates tested, and PCR primers designed from the T36 capsid protein gene sequence amplified successfully from all isolates. Based on these data, we propose the creation of two groups of CTV, exemplified by the VT and T36 isolates, respectively. Isolates in the VT group, which include isolates VT, T3, and T30, have genomic sequence divergence that is relatively constant in proportion and distribution throughout the genome, and candidate isolates for that group could be considered strains of the same virus. The T36 group is differentiated from the VT group by the highly divergent 5' genomic sequence. This 5' region of the CTV genome, thus, can serve as a measure of the extent of sequence divergence and can be used to define new groups and group members in the CTV complex.     

Nucleotide Sequence of Citrus Tristeza Virus Seedling Yellows Isolate

The complete nucleotide sequence of a seedling-yellows-inducing isolate NUagA of Citrus tristeza virus (CTV) was determined. It consisted of 19302 nucleotides and contained 12 open reading frames (ORF) organized identically to those of previously sequenced isolates. This genome is the largest among the CTV genome sequenced so far ; it is 6 nucleotides (nt), 76 nt, 43 nt, and 53 nt longer than that of T36 (quick decline, Florida), VT (seedling yellows, Israel), T385 (mild, Spain), and SY568 (stem pitting, California), respectively. Sequence comparison of NUagA and the other isolates revealed approximately 90% identities throughout the 3′ half of the genome. The 5′ half of the genome was only about 70% identical to that of T36 but still high at about 90% to those of VT, SY568, and T385. Comparison of amino acid sequences on ORF1a encoding polyproteins, the most variable region, reflects the CTV isolate relationship ; NUagA is closely related to VT, SY568, and T385, but distantly related to T36. Received 29 May 2000/ Accepted in revised form 16 November 2000     

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 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.     

Molecular analysis suggests that recent <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> outbreaks in Italy were originated by at least two independent introductions

Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. Numerous CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Italy in several citrus crops from three separate areas: (1) Cassibile, province of Syracuse; (2) Massafra, province of Taranto; and (3) Belpasso, province of Catania. CTV isolates from Massafra and Cassibile were mild, whereas isolates from Belpasso induced severe symptoms. To study the genetic variation of CTV populations of these areas, 150 samples per area were examined by single strand conformation polymorphism (SSCP) and nucleotide sequence analysis of CTV gene p20. All isolates from the same area showed the same SSCP pattern whereas for each area a different SSCP pattern was obtained. The Massafra and the Cassibile isolates had a nucleotide identity higher than 99% with a mild isolate from Spain and about 92% with the Belpasso isolates, which were similar (identity higher than 99%) to severe isolates from California and Japan. These results suggest at least two independent introductions of CTV in Italy, probably by import of CTV-infected budwoods. Within each area, the virus population was homogeneous suggesting diffusion of CTV by aphid transmission. The GenBank accession numbers of the sequences reported in this paper are: AY262000, AY263360 and AY263361 corresponding to gene p20 of CTV isolates from Massafra, Cassibile and Belpasso (Italy), respectively.     

应用改进的多克隆抗体Western blot技术研究柑桔速衰病毒蛋白

 本文利用多克隆抗体发展了一种无背景的Western blot技术并用于研究柑桔速衰病毒(CTV)的蛋白。结果表明,利用CTV兔多克隆抗体1212和1052发展的Western blot技术可以检测到感染CTV的墨西哥酸橙或Citrus excelsa植株内CTV的4种蛋白P1、P2、P3和P4。在健株或病株内杂交反应均无非特异性背景。从不同寄主上分离到的CTV不同分离物的蛋白条带是不同的。利用1212和1052抗体均可以检测到感染6个CTV分离物的墨西哥酸橙幼苗内的P1、P2和P3。利用1052抗体能检测到感染严重型分离物T36、T3和Mm2的墨西哥酸橙幼苗内微弱的P4,但感染轻型分离物T30、T26和T4的幼苗内则检测不到。利用1212抗体检测不到P4。在C. excelsa内,1212和1052抗体均能检测到感染所有分离物的病株内的P1。在感染T3、T26、T4或Mm2的病株内能检测到P2,但在感染T30和36分离物的病株内则检测不到。在感染T36、T3、T26、T4和Mm2的病株内可检测到P3,但在感染T30的病株内则检测不到。在大多数植物内,P1、P2、P3和P4的分子量分别约为25kDa,24kDa,21kDa和18kDa。在感染T36分离物的C. excelsa植株体内,P1和P3的分子量分别约为27kDa和22kDa,比感染其它分离物的C. excelsa和墨西哥酸橙内的P1和P3分子量略大。P1、P2和P3的分子量与利用单克隆抗体检测的CP,CP1和CP2的分子量相等。因此,P1、P2和P3可能是CTV的外壳蛋白CP,CP1和CP2。P4的特性不清楚。研究结果也表明,利用特异性的多克隆抗体进行的Western blot是研究CTV的一种有用的技术。应用该技术,病株内不同的CTV分离物或株系就可以通过特异性蛋白条带区分开来。     

First detection of a virulent strain of Citrus tristeza virus (Closteroviridae) in a citrus orchard of Chlef Valley (Algeria)

A large‐scale survey of Citrus tristeza virus (CTV) was carried out from 2016 to 2018 in the Chlef Valley, one of the main citrus growing areas in Algeria. In this study a total of 1680 citrus trees from 93 commercial orchards were sampled. The collected samples were tested by direct tissue blot immunoassay analysis and by the double antibody sandwich enzyme‐linked immunosorbent assay technique, and 54 trees were identified as being infected with CTV. This result confirmed that 54 trees were infected by the virus, corresponding to an infection rate of 3.21% throughout the studied area. Five of these local CTV sources were chosen for further molecular investigations to determine the genotype associated with the CTV isolates now spreading in the Chlef area. Characterization with multiple molecular markers showed the presence of the T30 and VT genotypes. This result allowed confirmation of the presence of a virulent strain belonging to the VT genotype. The other CTV isolates were similar to those from the Mitidja region, which showed 99% nucleotide identity with the Spanish mild CTV isolate. This early finding of a strain belonging to the VT genotype is an issue for Algerian citrus producers and needs rapid actions to be taken by the National phytosanitary services, extending the surveillance to other citrus production regions and uprooting the infected trees.     

Survey of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> (CTV) diversity in pigmented <Emphasis Type="Italic">Citrus x paradisi</Emphasis> (Macfad.) (Grapefruit) trees in north-western Argentina

Citrus tristeza virus (CTV) is the most severe viral pathogen of citrus and elicits a wide range of devastating disease symptoms. Grapefruit cultivars (Citrus x paradisi) are the most sensitive among citrus to the effects of CTV infections. Grapefruit is an important crop within the north-western Argentine citrus industry; however, production has been affected by CTV stem-pitting. In general, CTV diversity within South America is poorly studied, with data on grapefruit CTV populations being particularly limited. In this study, 50 samples were collected from Star Ruby, Henninger’s Ruby and Ruben Pink cultivars, within the provinces of Tucumán, Salta and Jujuy in north-western Argentina. The CTV p33 gene was PCR amplified and the resulting amplicons sequenced with Sanger sequencing. A subset of these amplicons was sequenced with Illumina MiSeq sequencing. AT-1-like sequences were dominant within the majority of populations, as determined by Sanger sequencing, followed by sequences clustering within the unresolved Kpg3/SP/T3 and RB clades. Sequencing by Illumina MiSeq confirmed this, as well as detecting minor sequence types within the HA 16–5, VT, B165 and A18 clades.     

Molecular variability of the 5'- and 3'-terminal regions of citrus tristeza virus RNA

ABSTRACT Isolates of citrus tristeza virus (CTV) differ widely in their biological properties. These properties may depend on the structure of viral RNA populations comprising the different isolates. As a first approach to study the molecular basis of the biological variability, we have compared the sequences of multiple cDNA clones of the two terminal regions of the RNA from different CTV isolates. The polymorphism of the 5' untranslated region (UTR) allowed the classification of the sequences into three groups, with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. The variability of an open reading frame (ORF) 1a segment adjacent to the 5' UTR supports the same grouping. Some CTV isolates contained sequences of more than one group. Most sequences from Spanish isolates belonged to group III, whereas a Japanese isolate was composed mostly of sequences of groups I and II. The mildest isolates contained only sequences of group III, whereas the most severe isolates also contained sequences of groups I, II, or both. The most stable secondary structure predicted for the 5' UTR was composed of two stem-loops and remained essentially unchanged as a result of compensatory mutations in the stems and accommodation of most of the variability in the loops. In contrast to the 5'-terminal region, the variability of the 3'-terminal region of CTV RNA was very much restricted, with nucleotide identity values higher than 90%. The presence of a conserved putative "zinc-finger" domain adjacent to a basic region in p23, the predicted product of ORF 11, suggests that this protein might act as a regulatory factor during virus replication.     

East Adriatic—a reservoir region of severe <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> strains

Citrus tristeza virus (CTV) represents one of the major threats to citrus production worldwide. In the East Adriatic region, CTV symptoms are mostly absent due to traditional citrus grafting on trifoliate orange (Poncirus trifoliata), a CTV-tolerant rootstock. Therefore, the virus has been continuously spreading by the propagation of infected material. The genetic variability of CTV was studied on nineteen citrus samples, collected from orchards in the coastal region of Croatia, Montenegro and Albania, that previously tested positive by ELISA and immunocapture RT-PCR. Single-strand conformation polymorphism of the amplified coat protein gene demonstrated the presence of different CTV variants in each amplicon, while sequence analysis of cloned CP gene variants confirmed their clustering into six out of the seven phylogenetic groups so far delineated. Four of these groups include sequences of severe quick decline, seedling yellows and stem-pitting (SP) isolates, thought to be found only rarely in the Mediterranean region. Regardless of the lack of symptoms in the field, CTV isolates from the East Adriatic displayed high genetic variability and pathogenic potential, additionally confirmed by biological characterisation. The high percentage of mixed infections suggest the potential for further diversification and a greater risk of severe variants spreading into new areas.     

Aphid Transmission Alters the Genomic and Defective RNA Populations of Citrus tristeza virus Isolates

ABSTRACT A total of 14 Spanish isolates of Citrus tristeza virus (CTV) and 1 isolate from Japan were transmitted by Aphis gossypii, and the subisolates obtained were compared with the source isolates for symptom expression and double-stranded RNA (dsRNA) pattern. Of the 14 Spanish isolates, 9 showed altered dsRNA patterns after aphid transmission but only minor variations in the intensity of symptoms induced on Mexican lime. Northern blot hybridization with complementary DNA (cDNA) probes corresponding to both the 5' and the 3' termini of the CTV genomic RNA (gRNA) showed that the dsRNA bands that could be used to discriminate between the dsRNA pattern of the source and the aphid-transmitted isolates were the replicative forms of defective RNAs (D-RNAs). Conversely, the Japanese isolate and two subisolates obtained from it by aphid transmission had the same dsRNA pattern, but one of the subisolates induced milder symptoms in several hosts. Dot-blot hybridization with cDNA probes representing several regions of the gRNA showed that most of the aphid-transmitted isolates differed from the corresponding source isolate by their hybridization pattern. Our results indicate that aphid transmission often sorts the populations of gRNA variants and D-RNAs present in CTV isolates.     

Characterization of Citrus tristeza virus isolates in northern Iran

The biological and molecular properties of four Citrus tristeza virus (CTV) isolates isolated from infected Satsuma trees imported from Japan, and growing in citrus groves in northern Iran (Mahdasht orchards, Mazandaran Province), were investigated. CTV-infected samples were collected from sweet orange trees and grafted onto Alemow (Citrus macrophylla Wester) seedlings. On indicator plants, these isolates produced various symptoms including vein clearing and stem pitting on Mexican lime, Alemow, and Citrus hystrix, and yellowing and stunting on sour orange and grapefruit seedlings. Citrus samples were also surveyed for CTV using serological tests. The coat protein (CP) gene of these isolates was amplified using specific primers, yielding an amplicon of 672 bp for all isolates. Sequence analysis showed 98%–99% sequence homology of Iranian isolates with the Californian CTV severe stem-pitting isolate SY568 and 97%–98% homology with the Japanese seedling yellows isolate NUagA. The Iranian isolates were compared by restriction fragment length polymorphism (RFLP) analysis of the CP amplicon for further classification.     

Diversity of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> populations in commercial grapefruit orchards in Southern Africa,determined using Illumina MiSeq technology

Grapefruit cultivars are highly sensitive to CTV infections and in order to increase their productive lifespan, the Southern African citrus industry makes use of cross-protection. However, the breakdown of cross-protection is commonly observed in commercial grapefruit plantings. In order to determine which genotypes of CTV are associated with commercial Citrus x paradisi (Macfad.) cv. ‘Star Ruby’ in Southern Africa, 192 samples, pre-immunised with the GFMS 12 population, were collected from the grapefruit production areas of Hoedspruit, Malelane, Swaziland, Northern Cape, Sundays River Valley and Nkwalini Valley and six samples from non-pre-immunised plants in Letsitele as well as three samples from greenhouse maintained plants harbouring populations derived from the original GFMS 12 source. The p33 gene was amplified with direct Sanger sequencing performed on the resulting amplicons. A subset of 92 samples randomly selected and p33 gene amplicons subjected to Illumina MiSeq amplicon sequencing. High levels of CTV diversity were observed between and within orchards. Most populations were made up of a dominant component with several minor sequence types. Resistance Breaking (RB) sequences were most numerous, especially in recently planted orchards and present within all of the populations. The Kpg3/SP/T3 group appeared to be the second most prevalent, with increased prevalence in older orchards. Sequences mapping to HA 16–5, VT, AT-1, T36, Taiwan-Pum/M/T5 and T30, were represented sporadically within numerous collection sites.     

Selective transmission of the seedling yellows-type genotypic variants of Citrus tristeza virus by Aphis gossypii in northern Iran

Citrus tristeza virus (CTV) has existed in northern Iran for more than five decades. The long-time interaction of different virus genotypes with Aphis gossypii, as the only aphid vector of CTV in northern Iran, has led to the emergence of highly virulent subpopulations, among others, in the established foci. Here, we studied the population structure of the originally established CTV isolates present in Satsuma mandarin (Citrus unshiu) trees imported from Japan, and subisolates thereof, formed following experimental transmission by A. gossypii, as well as those evolved through natural transmission by this aphid species in the groves. Symptoms of the naturally spread and the experimentally aphid-transmitted isolates were similar to those of the Satsuma CTV source isolates for all indicator plants except for sour orange (Citrus aurantium) and grapefruit (Citrus paradisi), with the aphid-transmitted isolates additionally inducing severe seedling yellows and stunting in these two indicators. Studies on the population heterogeneity of these isolates through comparison of their single-strand conformational polymorphism profiles and nucleotide sequences of the 25 kDa capsid protein gene from the predominant haplotypes, and dot-blot hybridization signals, revealed the presence of two major T36- and SY568- (or NUagA-) like genotypes along with a minor poorly characterized one in the originally infected Satsuma trees; in contrast, only a certain genomic variant having the highest similarity to the isolate SY568 (and NUagA) was predominant both in the naturally infected trees and in those infected experimentally by A. gossypii. It seems that transmission by A. gossypii to sweet orange (Citrus sinensis) has led to the preponderance of the CTV genomic variants inducing severe seedling yellows in northern Iran.     

Discriminating microsatellites from Macrophomina phaseolina and their potential association to biological functions

One hundred and eighty‐two microsatellites or simple‐sequence‐repeat (SSR) markers for Macrophomina phaseolina were developed. These were tested on 24 isolates of M. phaseolina obtained from seven plant species, and the genetic variation of isolates was studied in relation to potential biological processes that could be affected in this fungus. A total of 120 SSR markers were polymorphic, amplifying >90% of the 24 isolates tested. Thirty percent of the markers showed multiple alleles on individual samples. A large number of markers showed unique alleles in isolates collected from pumpkin and snap bean. DNA sequences corresponding to 43 markers had significant hits on blast x and/or blast2go , and the polymorphism of 36 of those markers showed specific allele patterns for one or more plant host origin of the isolates. Additional tests on growth rate and copper resistance of the isolates identified markers that could be related to those traits. In addition, 27 markers were monomorphic and amplified all 24 isolates. Whereas polymorphic markers can be used for population genetics studies of M. phaseolina, the group of 27 monomorphic markers could help in the fast identification of this species in clinical specimens. The SSR markers developed here will enrich the limited molecular marker resource in M. phaseolina and could be used as the basis for more in‐depth studies of the host‐pathogen interactions of M. phaseolina.     

广西柑橘衰退病和黄脉病调查及其病原病毒的遗传多样性分析

为明确柑橘衰退病毒(citrus tristeza virus, CTV)和柑橘黄脉病毒(citrus yellow vein clearing virus, CYVCV)在广西柑橘上的发生?分布及其遗传变异情况, 于2020年至2021年对百色?北海?崇左?贵港?桂林?河池?贺州?来宾?柳州?南宁?梧州和玉林等12个柑橘产区进行了病毒病调查?采用RT-PCR对采集样品进行了病毒检测, 并基于病毒分离物外壳蛋白(coat protein, CP)基因的核苷酸序列进行比对分析, 构建系统发育树?结果表明:采集的737份柑橘样品中, CTV的检出率为20.62%, CYVCV检出率为18.32%, CTV的检出率略高于CYVCV?病毒复合侵染的现象在采集的柑橘样品中普遍存在, CTV和CYVCV复合侵染率高达34.50%?对RT-PCR产物测序共获得12个CTV分离物和6个CYVCV分离物的CP基因序列?遗传多样性分析发现, CTV和CYVCV的CP基因序列都较保守, CTV分离物的遗传进化与地理来源?寄主来源均没有明显相关性, 但CYVCV分离物的遗传进化与地理位置具有相关性, 而与寄主来源无明显相关性?上述研究结果可为深入了解CTV和CYVCV在广西的流行情况以及柑橘病毒病的检疫和防控提供参考?     

瓜类褪绿黄化病毒山东分离物全基因组序列扩增及分析

为明确分离自山东省寿光市甜瓜上的瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)分离物的全基因组序列信息和遗传变异情况,利用毛形病毒属Crinivirus简并引物进行RTPCR检测,利用RACE技术结合RT-PCR方法克隆CCYV山东分离物2条RNA链的全基因组序列,通过与GenBank中其它地区CCYV分离物的全长序列进行比对分析其同源性,并基于CP基因序列构建系统进化树分析其遗传变异情况。结果表明,山东分离物经RT-PCR检测和测序后确定为CCYV。CCYV山东分离物与其它CCYV分离物的RNA1链和RNA2链的全基因组序列一致性的平均值分别为99.82%和99.88%,且2条链的5′末端均比较保守,没有碱基突变的情况发生;RNA1链3′末端存在2个碱基变异,RNA2链3′末端存在1个碱基变异。CCYV不同地区分离物主要分为3个簇群,其中山东分离物和中国其它地区分离物、日本分离物、苏丹分离物、黎巴嫩分离物和塞浦路斯分离物聚类在一起。研究表明CCYV基因组序列比较保守,该病毒的分化可能与地理来源存在一定的相关性。     

湖南柑橘衰退病调查分析及弱毒系筛选

 The investigation showed that stem-pitting Citrus tristeza virus (CTV)occurred commonly in citrus production areas in several varieties of Hunan Province. Accurate detection of CTV strains was performed by p23/PCR method, PCR and the results indicated that the most samples were infected with several CTV isolates. Three mild strains were isolated and their pathogenicity was identified by biological identification, it indicated that p23/PCR groups had uniformity with the pathogenicity of CTV isolates. Furthermore, three mild isolates were tested in the cross protection by analysis of biological symptoms and composition of p23 gene. Different protecting effects were observed among these strains and W17 mild isolate was effective.     

Development,characterization and application of genomic SSR markers for the oat stem rust pathogen Puccinia graminis f. sp. avenae

Oat stem rust, caused by Puccinia graminis f. sp. avenae (Pga), is one of the most severe diseases of oats worldwide. Population studies are scarce for this pathogen, mainly due to the lack of polymorphic molecular markers suitable for genetic analysis. In this study, an Australian Pga isolate was sequenced, the abundance of simple sequence repeats (SSRs) was determined and PCR‐based polymorphic markers suitable for genetic diversity analysis were developed. The amplification of 194 primer pairs was initially assessed using a set of 12 isolates of different cereal rust species and their formae speciales. A high frequency of cross‐species amplification was observed for most markers; however, 36 SSRs were diagnostic for P. graminis only. A subset of 19 genome‐derived SSRs were deemed useful for genetic diversity analysis of Pga and were assessed on 66 Pga isolates from Australia, Brazil and Sweden. Brazilian and Australian isolates were characterized by one and two predominant clonal lineages, respectively. In contrast, the Swedish isolates, previously shown to undergo sexual recombination, were highly diverse (nine distinct genotypes out of 10 isolates) and divided into two subpopulations. The genome‐derived SSR markers developed in this study were well suited to the population studies undertaken, and have diagnostic capabilities that should aid in the identification of unknown rust pathogen species.     

Comparison of the minor coat protein gene sequences of aphid-transmissible and -nontransmissible isolates of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis>

The nucleotide sequences for the minor coat protein (CPm) gene and its deduced amino acid sequences for two aphid-transmissible and two nontransmissible isolates of Citrus tristeza virus (CTV) from symptomless orchard trees of Miyagawa satsuma [Citrus unshiu (Macf.) Marc.] on trifoliate orange [Poncirus trifoliate (L.) Raf.] and declining Washington navel [C. sinensis (L.) Osb.] trees on sour orange (C. aurantium L.) rootstocks were analyzed and compared with those of highly transmissible CTV strains available in GenBank. The isolates produced severe symptoms on indicator plants and their aphid transmissibility was assayed through acquisition by A. gossypii of CTV and subsequent inoculation feeding on young Mexican lime seedlings. The CPm gene nucleotides and coded amino acid sequences were very similar among the nontransmissible isolates and among the transmissible. Five of 73 nucleotide substitutions that existed between CPm gene nucleotide sequence of nontransmissible and transmissible isolates caused changes in the deduced amino acid sequences of the nontransmissible isolates. Two nucleotide substitutions yielded new amino acids with similar properties. However, the three remaining mutations led to substitution of new amino acids with a different charge and polarity at positions 14, 238 and 239. The last two mutations occurred at the C-terminal region of the CPm, which is implicated in the formation of a salt bridge that helps to maintain the protein’s tertiary structure. Amino acid substitutions can affect aphid transmission efficiency by altering the conformation of the proteins or masking motifs involved in the interaction between CPm and aphid stylets.