Characterization of the first two viruses described from wild populations of hammer orchids (Drakaea spp.) in Australia

Sequences representing the genomes of two distinct virus isolates infecting wild plants of two members of the genus Drakaea (hammer orchids) in Western Australia are described. The virus isolated from Drakaea livida has a bipartite genome of 4490 nt (RNA1) and 2905 nt (RNA2) that shares closest sequence and structural similarity to members of the genus Pecluvirus, family Virgaviridae, described from legumes in the Indian subcontinent and West Africa. However, it differs from pecluviruses by lacking a P39 protein on RNA2 and having a cysteine‐rich protein gene located 3′ of the triple gene block protein genes. It is the first peclu‐like virus to be described from Australia. The name Drakaea virus A is proposed (DVA; proposed member of the family Virgaviridae, genus unassigned). The second virus isolate was identified from Drakaea elastica, a species classed as endangered under conservation legislation. The genome sequence of this virus shares closest identity with isolates of Donkey orchid symptomless virus (DOSV; proposed member of the order Tymovirales, family and genus unassigned), a species described previously from wild Caladenia and Diuris orchids in the same region. These viruses are the first to be isolated from wild Drakaea populations and are proposed to have an ancient association with their orchid hosts.     

Genetic variation and evolutionary forces shaping Cucumber vein yellowing virus populations: risk of emergence of virulent isolates in Europe

The genetic variation and evolutionary mechanisms shaping Cucumber vein yellowing virus (CVYV) populations were investigated by analysis of nucleotide sequences coding for P1b, P1b/P3 and coat proteins (CP) from isolates collected in different countries. The complete genome sequence of isolate ISM from Israel was also determined and compared to those of isolates Jor from Jordan and ALM32 from Spain. This isolate had overall nucleotide identities of 94·23 and 94·96% with ALM32 and Jor, respectively. Nucleotide variation among isolates was not homogeneously distributed, with the 5′ half of the genome being more variable than the 3′ half. A Bayesian phylogenetic tree of the CP showed that CVYV isolates clustered into two main clades: isolates from the Middle East region (Lebanon, Israel and Jordan) clustered in both clades whereas the isolate from Tunisia clustered in clade I and the European isolates clustered as a homogeneous phylogroup in Clade II. A similar topology was observed for P1b but with incongruences with respect to the CP, suggesting genetic exchange among virus isolates, which were confirmed with recombination algorithms. The low genetic diversity within the European phylogroup with respect to the other isolates, neutralist tests and genetic differentiation analyses suggest that the Middle East region is the cradle of CVYV and that a unique virus introduction event occurred in Europe, where the virus spread rapidly. Taken together, these findings indicate a risk of emergence of virulent CVYV isolates in Europe either through migration from the Middle East or by genetic changes of the European isolates.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454     

Detection of Tomato yellow leaf curl virus in imported tomato fruit in northern Europe

Imported tomato fruits infected with Tomato yellow leaf curl virus (TYLCV) were identified on the market in northern Europe using paper‐based FTA Classic Cards (Whatman), polymerase chain reaction (PCR) and partial DNA sequence analysis. Trade tomatoes originating from southern Europe, Africa and the Middle East were sampled in Estonia and Sweden, and tested for infection with begomoviruses. Out of 100 batches analysed with five fruits sampled in each batch (58 batches from Estonia and 42 from Sweden), 20 batches were positive (16 from Estonia and four from Sweden). Rolling circle amplification (RCA) and full‐length genome sequence analysis of one isolate collected in Estonia and one isolate in Sweden, revealed highest nucleotide sequence identity at 99% to TYLCV‐IL for the Estonian isolate and at 97% to TYLCV‐Mld for the Swedish isolate. In this study, TYLCV was identified for the first time in imported tomato fruits on the market in northern Europe. FTA cards proved to be an effective means to collect, extract and store begomovirus DNA from tomato fruits and the subsequent molecular analysis.     

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.     

水蜡A病毒在呼和浩特市和哈尔滨市紫丁香上的发生及其基因组分子特征分析

为明确侵染紫丁香Syringa oblata并引起褪绿花叶症状的病毒种类及其基因组分子特征,利用透射电子显微镜对分离自呼和浩特市和哈尔滨市的紫丁香病样中的病毒粒子进行观察,并通过小RNA高通量测序和RT-PCR技术对其进行检测分析。结果表明,在紫丁香显症叶片的病毒粗提液中观察到长约600 nm、宽约13 nm的线状病毒粒子。利用小RNA高通量测序和RT-PCR技术从病样中检测到水蜡A病毒（Ligustrum virus A,LVA）,发病率为3.7%。呼和浩特市紫丁香分离物LVA-Sob的基因组序列全长8 525 nt,包含6个开放阅读框,分别编码Rep（1 968 aa）、TGB1（229 aa）、TGB2（107 aa）、TGB3（60 aa）、CP（294 aa）和NABP（119 aa）共6个蛋白。序列一致性分析表明,分离物LVA-Sob与韩国水蜡树分离物LVA-SK的基因组序列一致率高达97.9%,而与我国辽宁省暴马丁香分离物LVA-DX的基因组序列一致率仅为73.6%。在这3个LVA分离物基因组中没有检测到重组事件;基于基因组和cp基因序列的系统发育树显示这3个LVA分离物形成一个分支,并与瑞香S病毒（daphne virus S,DVS）有较近的亲缘关系。     

黄瓜花叶病毒两个甜瓜分离物的基因组及其侵染性克隆

为明确黄瓜花叶病毒（cucumber mosaic virus,CMV）甜瓜分离物的分子变异情况及其侵染性,对2个甜瓜分离物CH99和XH18的基因组进行克隆、测序和分析,并通过构建全长cDNA克隆分析其侵染性。结果显示,黄瓜花叶病毒甜瓜CH99分离物3条RNA长度分别为3 356、3 049和2 211 nt,甜瓜XH18分离物3条RNA长度分别为3 381、3 048和2 217 nt。分离物CH99与XH18的核苷酸序列一致性为89.40%～95.80%,氨基酸序列一致性为90.00%～97.80%,CH99分离物与其他CMV分离物的核苷酸和氨基酸序列一致性平均值分别为79.23%～89.29%和73.52%～93.90%,XH18分离物与其他CMV分离物的核苷酸和氨基酸序列一致性平均值分别为79.81%～89.83%和74.02%～95.14%。遗传发育分析显示,这2个分离物均属于亚组IB成员。接种试验结果显示,分离物CH99和XH18的侵染性克隆构建成功,这2个分离物均能系统侵染本生烟、甜瓜和黄瓜,并在本生烟和甜瓜上引起较严重的症状,在黄瓜上引起的症状较弱,而二者均不能侵染西...     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

油桃茎痘相关病毒中国分离物基因组的分子特征分析

为明确我国油桃茎痘相关病毒(nectarine stem-pitting-associated virus,NSPaV)基因组的分子特征,利用RT-PCR和RACE技术对NSPaV中国分离物NSPaV-T04基因组进行克隆,采用最大似然法对得到的NSPaV基因组序列和GenBank中的5条NSPaV基因组序列构建系统发育树,应用RDP软件对NSPaV基因组序列进行重组分析。结果表明:中国分离物NSPaV-T04基因组序列全长为4 991 nt,包括4个开放阅读框(open reading frame,ORF),其中ORF1与ORF2共同编码1个RdRp P1-P2融合蛋白,ORF3编码1个CP,ORF5与ORF3共同编码CP通读蛋白。系统发育树和序列比较分析结果显示,中国分离物NSPaV-T04(MN095353)与美国分离物NSPaV/12P42(KT273410)的亲缘关系最近,核苷酸序列同源性最高,为96.4%;NSPaV-T04的RdRp P1变异较大,与GenBank中5条NSPaV基因组核苷酸序列的同源性为90.5%~96.1%,CP较为保守,核苷酸序列的同源性为96.6%~98.7%。重组分析结果显示,中国分离物NSPaV-T04为鉴定的一个重组体(韩国分离物SK)的亲本序列,表明中国分离物NSPaV-T04可能是一个实际的重组体。     

甘薯病毒G CH株系和CH2株系中国分离物基因组全序列克隆及结构特征分析

为明确甘薯病毒G(sweet potato virus G,SPVG)CH株系中国分离物SPVG-CH-Ch1和CH2株系中国分离物SPVG-CH2-Ch1的基因组结构特征及遗传变异情况,利用RT-PCR和RACE方法克隆获得分离物SPVG-CH-Ch1和SPVG-CH2-Ch1的基因组全序列,应用DNAMAN软件对基因组全序列及不同编码区序列进行分子变异分析,并基于多聚蛋白基因序列利用MEGA 7软件进行系统进化分析。结果表明,分离物SPVG-CH-Ch1和SPVG-CH2-Ch1的基因组分别包含10 813个和10 834个核苷酸,均包含1个开放阅读框,由10 467个核苷酸组成,编码含有3 488个氨基酸残基的多聚蛋白。分离物SPVG-CH-Ch1与SPVG-CH2-Ch1之间的基因组全序列核苷酸一致性为78.6%,二者与GenBank中登录的其它SPVG分离物基因组全序列核苷酸一致性分别为78.6%～99.1%和77.9%～98.6%,其中SPVG-CH-Ch1与IS103分离物(KM014815)的核苷酸一致性最高,为99.1%,与WT325分离物(KF790759)的核苷酸...     

Species composition,toxigenic potential and pathogenicity of Fusarium graminearum species complex isolates from southern Brazilian rice

This study aimed to assess the extent and distribution of Fusarium graminearum species complex (FGSC) diversity in rice seeds produced in southern Brazil. Four species and two trichothecene genotypes were detected among 89 FGSC isolates, based on a multilocus genotyping assay: F. asiaticum (69·6%) with the nivalenol (NIV) genotype, F. graminearum (14·6%) with the 15‐acetyldeoxynivalenol (ADON) genotype, and F. cortaderiae (14·6%) and F. meridionale (1·1%), both with the NIV genotype. Seven selected F. asiaticum isolates from rice produced NIV in rice‐based substrate in vitro, at levels ranging from 4·7 to 84·1 μg g^?1. Similarly, two F. graminearum isolates from rice produced mainly 15‐ADON (c. 15–41 μg g^?1) and a smaller amount of 3‐ADON (c. 6–12 μg g^?1). One F. meridionale and two F. cortaderiae isolates did not produce detectable levels of trichothecenes. Two F. asiaticum isolates from rice and two from wheat (from a previous study), and one F. graminearum isolate from wheat, were pathogenic to both crops at various levels of aggressiveness based on measures of disease severity in wheat spikes and rice kernel infection in a greenhouse assay. Fusarium asiaticum and the reference F. graminearum isolate from wheat produced NIV, and deoxynivalenol and acetylates, respectively, in the kernels of inoculated wheat heads. No trichothecene was produced in kernels from inoculated rice panicles by any of the isolates. These findings constitute the first report of FGSC composition in rice outside Asia, and confirm the dominance of F. asiaticum in rice agroecosystems.     

A Tobacco etch virus NW isolate that overcomes pvr1 and pvr12 resistance in Capsicum sp.

Two important sources of Capsicum annuum (bell pepper) resistance were evaluated for their response to inoculation with two isolates of Tobacco etch virus strain NW (TEV‐NW, genus Potyvirus). The resistant cultivars were CA4 and Dempsey, which contain the pvr1 and pvr1² resistance genes, respectively. TEV‐NW was maintained by mechanical passage in the susceptible pepper cultivar Early Calwonder and Nicotiana tabacum cv. Kentucky 14. In initial experiments, the TEV‐NW isolate maintained in Early Calwonder infected two of seven CA4 plants; however, none of the CA4 plants inoculated with the TEV‐NW isolate maintained in Kentucky 14 were infected. The infected CA4 plants had low virus titres in non‐inoculated leaves and did not develop visible symptoms. When the infected CA4 plants were used as inoculum of additional CA4 plants, all newly inoculated plants became infected, developed systemic symptoms and accumulated virus in non‐inoculated leaves more quickly than the originally infected CA4 plants. This new NW isolate, referred to as NW‐CA4, was shown to overcome the resistances expressed by both CA4 (pvr1) and Dempsey (pvr1²). The potyviral VPg is believed to be the determinant for pvr1 and pvr1² resistance genes, both of which are eIF4E‐encoding genes. The VPg amino acid sequence for NW‐CA4 was determined and compared with that of NW isolates and different TEV strains. No amino acid variation was identified that explained the infectivity of NW‐CA4 in CA4 and Dempsey plants.     

猕猴桃褪绿环斑相关病毒在陕西省的分布、分子变异及其2个分离物的基因组序列

为明确我国陕西省猕猴桃主产区的徐香、海沃德、华优和秦美4个猕猴桃品种上的猕猴桃褪绿环斑相关病毒(Actinidia chlorotic ringspot-associated virus,AcCRaV)的分布情况,对采集自该省4个地区的493份样品进行AcCRaV检测,基于cp基因序列对获得的AcCRaV分离物进行分子变异分析,并采用高通量测序技术对其中2份样品进行转录组测序。结果显示:AcCRaV在陕西省猕猴桃上分布广泛,且检出率较高,其中周至县秦美猕猴桃上AcCRaV的检出率最高,为45.0%,在杨凌区秦美猕猴桃上AcCRaV的检出率最低,为10.0%。测定的23个AcCRaV分离物cp基因序列全长均为945 nt。系统发育树显示AcCRaV分离物共分成2个组,存在较大的分子变异。AcCRaV的cp基因分子变异与猕猴桃品种有一定关系,而与地理位置相关性不明显。获得了2个AcCRaV分离物ZZ1和ZZ2的基因组序列,RNA1长度分别为7 049 nt和7 274 nt,RNA2长度均为2 266 nt,RNA3长度分别为1 691 nt和1 696 nt,RNA4长度分别为1 736 nt和1 683 nt,RNA5长度分别为1 460 nt和1 497 nt。分离物ZZ1和ZZ2的基因组序列与GenBank中唯一报道的我国湖北省AcCRaV分离物HN-6基因组序列比对中,分离物ZZ2与HN-6的RNA4同源率最高,为96.0%,分离物ZZ2与HN-6的RNA1同源率最低,为87.8%。表明AcCRaV在我国陕西省猕猴桃主产区分布较广泛,且其分子变异与猕猴桃品种有一定关系。     

Occurrence and diversity of Tomato spotted wilt virus isolates breaking the Tsw resistance gene of Capsicum chinense in Yunnan,southwest China

Widely used resistant peppers (Capsicum spp.) bearing the Tsw locus triggered the rapid emergence of resistance‐breaking (RB) isolates of Tomato spotted wilt virus (TSWV) around the world. However, although TSWV‐induced diseases have rapidly increased in Yunnan, southwest China, in recent years, no information is available about the diversity of TSWV isolates in this region. In this study, the occurrence of natural TSWV RB variants among isolates collected in Yunnan is reported. Initially, a TSWV isolate from asparagus lettuce (TSWV‐LE) was collected in Yunnan in 2012. Surprisingly, this isolate of TSWV induced systemic necrosis on pepper carrying the Tsw resistance gene. Novel TSWV isolates, collected in 2015, included a tomato isolate (TSWV‐YN18) and a tobacco isolate (TSWV‐YN53) that also overcame Tsw‐mediated resistance. TSWV‐YN18 induced systemic ringspots, whereas TSWV‐YN53 caused systemic chlorotic mottling. Variations in the TSWV nonstructural (NSs) protein are the key determinants associated with Tsw resistance‐breaking isolates. It was found that TSWV‐LE NSs retained the hypersensitive response (HR) induction, whereas TSWV‐YN18 and TSWV‐YN53 NSs were unable to induce HR. However, the NSs of all three RB isolates suppressed RNA silencing. Sequence analysis of the NSs revealed that RB isolates of Yunnan have no amino acid mutation sites common to other previously reported RB isolates. However, two amino acids (F74 and K272) on TSWV‐LE NSs make it distinct from TSWV‐YN18 and TSWV‐YN53. The occurrence of different RB isolates and the failure of Tsw‐mediated resistance control pose serious threats to domestic pepper crops in southwest China.     

Fusarium solani f. sp. passiflorae: a new forma specialis causing collar rot in yellow passion fruit

The aim of this study was to characterize a Fusarium population obtained from yellow passion fruit (YPF) with collar rot using pathogenicity, morphocultural characteristics and molecular tests. Pathogenicity and disease severity were assessed in six plant species: YPF, zucchini, tomato, bean, soya bean and cucumber. Potato dextrose agar medium (PDA) was used to determine mycelial growth at five temperatures (15–35°C). The colour produced by isolates was also determined on PDA at 25°C. Synthetic nutrient agar medium was used to evaluate: (i) type of mycelium and phialides; (ii) size, shape and number of septa from conidia; and (iii) production of chlamydospores and perithecia. Molecular tests consisted of sequencing the ITS–5·8S rDNA region and elongation factor 1α (EF‐1α) gene. The isolates caused large lesions on YPF, zucchini and tomato, with YPF having the highest mean disease severity and being the only one that showed wilt symptoms and death of the plant. Thus the isolates showed host specificity. Maximum mycelial growth occurred at 25°C and the predominant colour was bluish‐white. The isolates produced long phialides, dense aerial mycelium, oval microconidia with a mean size of 9·5 × 2·6 μm, macroconidia of 32·7 × 3·4 μm with 3·3 septa, and chlamydospores; only one isolate lacked perithecia. Phylogenetic trees of the ITS region and EF‐1α gene showed that isolates from YPF formed a distinct group within the F. solani group and the formae speciales of F. solani. It is proposed to name all isolates from YPF as F. solani f. sp. passiflorae.     

甘薯病毒C中国分离物全基因组序列测定及比较分析

利用RT-PCR结合RACE方法,从采自河南南阳的甘薯样品上获得甘薯病毒C中国分离物(SPVC-Ch1)的全长基因组序列。序列分析结果表明,SPVC-Ch1基因组由1 0846个核苷酸组成,3'末端包含poly(A)尾序。基因组含有1个由10 446个核苷酸构成的开放阅读框,编码一个由3 481个氨基酸残基构成的393 k Da多聚蛋白。将SPVC-Ch1与Gen Bank中登录的其他SPVC分离物序列进行比较分析发现,SPVC不同分离物间全基因组核苷酸序列相似性为92.7%~98.9%,多聚蛋白的氨基酸序列相似性为95.1%~99.2%,SPVC-Ch1与Bungo分离物的相似性最高,与C1分离物的相似性最低。系统进化树分析结果表明,SPVC-Ch1与日本的Bungo、以色列的IL、韩国的CW135和UN202等分离物形成一个分支,亲缘关系较近。这是SPVC中国分离物全基因组序列的首次报道,研究结果丰富了SPVC全基因组序列信息,有助于全面了解SPVC种群的遗传进化关系。     

Diversity and pathogenicity of Fusarium graminearum species complex from maize stalk and ear rot strains in northeast China

The Fusarium graminearum species complex (FGSC) is an important group of pathogens distributed in maize‐producing areas worldwide. This study investigated the genetic diversity and pathogenicity of 40 FGSC isolates obtained from stalk rot and ear rot samples collected from 42 locations in northeastern China during 2013 and 2014. A phylogenetic tree of translation elongation factor (EF‐la) sequences designated the 40 isolates as F. graminearum sensu stricto (67.5%) and F. boothii (32.5%). By using inter‐simple sequence repeat analysis (ISSR), it was shown that the isolates were divided into two clades, which corresponded to the species identity of the isolates. However, the isolates from the two different diseases could not be distinguished in pathogenicity. The disease severity index of seedlings inoculated with stalk isolates was slightly higher than that of seedlings inoculated with isolates from infected ears, whereas the pathogenicity of the stalk and ear isolates were identical.