Effect of temperature on RNA silencing of a negative‐stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative‐stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft‐inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV‐derived small RNAs (CPsV‐sRNAs) slightly increased at 32/26°C, with the ratio of CPsV‐sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.     

Transgenic Nicotiana occidentalis Plants Expressing the 50-kDa Protein of Apple chlorotic leaf spot virus Display Increased Susceptibility to Homologous Virus, but Strong Resistance to Grapevine berry inner necrosis virus

ABSTRACT The 50-kDa protein (P50) encoded by the open reading frame 2 of Apple chlorotic leaf spot virus (ACLSV), a putative movement protein, was expressed in transgenic Nicotiana occidentalis plants. P50 in transgenic plants was mainly detected in a modified form in the cell wall fraction, similar to that in infected leaves. The P50-expressing plants (P50 plants) complemented the systemic spread of the P50-defective mutants of an infectious cDNA clone of ACLSV (pCLSF), indicating that P50 in transgenic plants was functional. Severity of symptoms was greatly enhanced and accumulation of virus in upper leaves was increased in P50 plants inoculated with pCLSF or ACLSV compared with that in nontransgenic control plants (NT plants). Conversely, transgenic plants expressing the coat protein of ACLSV (CP plants) showed a significant delay in symptom development and a reduction of virus accumulation. However, most P50 plants inoculated with Grapevine berry inner necrosis virus (GINV), another species of the genus Trichovirus, neither developed obvious symptoms nor supported virus accumulation in inoculated or upper leaves. In contrast, systemic symptoms developed and virus accumulated equally in NT and CP plants inoculated with GINV. After inoculation with Apple stem grooving virus or Apple stem pitting virus, there was no difference in symptom development and virus accumulation among P50, CP, and NT plants. Our results indicate that transgenic plants expressing a functional P50 were more susceptible to homologous virus and, on the contrary, showed strong resistance to the heterologous virus GINV.     

苹果茎沟病毒柑橘碎叶株系的分布与序列分析

为明确苹果茎沟病毒(apple stem grooving virus,ASGV)柑橘碎叶株系(citrus tatter leaf virus,CTLV)在中国柑橘产区的发生分布及其分子特性,于2017—2021年对我国12个柑橘主产区开展系统调查,并采用多重比对以及系统发育分析法对CTLV的全序列进行分析。结果表明,从12个柑橘主产区采集的2 012份疑似样品中检测出413份感CTLV阳性样品。除陕西省外,其余11个柑橘主产区样品中均检测出CTLV,并且柚类样品中CTLV的检出率最高,达到32.31%。对分离获得的22株CTLV毒株和已知的7株CTLV以及5株ASGV毒株进行全序列分析,发现所有34株毒株的核苷酸序列相似性为78.6%～99.8%,且CTLV序列的保守性较高。此外,与其他4株ASGV毒株相比,本研究中22株CTLV毒株与ASGV-MK毒株(GenBank登录号为MZ127820.1)具有更近的亲缘关系。推测CTLV中国毒株间的亲缘关系可能与其采样地和寄主品种有关。     

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.     

Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, is the presumed causal agent of a bark scaling disease in citrus plants. CPsV virions are kinked filaments composed of three negative‐strand RNA molecules and a ～48‐kDa coat protein. The virus induces two different syndromes: psorosis A (PsA), characterized by limited bark scaling lesions in the trunk and main limbs, and a more aggressive form of the disease called psorosis B (PsB) with rampant bark lesions affecting even thin branches and chlorotic blotches in old leaves. In the greenhouse, the PsA and PsB syndromes can be induced by graft inoculating healthy citrus seedlings with non‐lesion or with lesion bark inoculum from PsA‐affected field trees. PsA‐ and PsB‐inducing CPsV sub‐isolates obtained by this procedure from the same tree showed identical single‐strand conformation polymorphism (SSCP) profiles in homologous segments of the RNAs 1 and 3, whereas segments of the RNA 2 enabled discrimination between PsA‐ and PsB‐associated sequence variants. SSCP analysis of the RNA 2 population present in different tissues of psorosis‐infected plants showed that: (i) PsA‐inducing isolates contain PsB‐associated sequence variants at low frequency, (ii) the PsB‐associated sequence variant is predominant in blistered twigs and gummy pustules affecting old leaves, characteristic of PsB isolates, and (iii) the PsB‐associated sequence variant accumulates preferentially in bark lesions of the trunk and limbs. SSCP analysis of the RNA 2 population also enabled monitoring of interference between PsA‐ and PsB‐associated variants in plants co‐inoculated with both psorosis types.     

北京月季病原病毒的高通量测序鉴定和RT-PCR检测

本研究利用高通量测序技术对北京地区的月季染病样品进行了病毒鉴定,通过序列比对和拼接获得了李属坏死环斑病毒(prunus necrotic ringspot virus,PNRSV)、苹果茎沟病毒(apple stem grooving virus,ASGV)、柑橘碎叶病毒(citrus tatter leaf viru...     

番茄黄化曲叶病毒北京分离物侵染性克隆的构建与其致病性

为明确番茄黄化曲叶病毒北京分离物（Beijing isolate of tomato yellow leaf curl virus,TYLCV-BJ）致病性的强弱,以感染TYLCV-BJ的番茄叶片DNA为模板PCR扩增获得该分离物基因组全长序列,并构建该分离物的侵染性克隆,将其分别接种到番茄、烟草和拟南芥植株上,比较该分离物和TYLCV上海分离物2(TYLCV-Shanghai 2,TYLCV-SH2)致病性的差异。结果显示,该分离物基因组全长序列同TYLCV-SH2的相似度为99.03%,在番茄和烟草植株上TYLCV-BJ比TYLCV-SH2发病更早,症状更重,TYLCV DNA和外壳蛋白积累量更高。TYLCV-BJ可以通过农杆菌Agrobacterium tumefaciens注射法在拟南芥中复制和系统侵染,而TYLCV-SH2不能有效侵染拟南芥。表明TYLCV-BJ的致病性强于TYLCV-SH2,所建立的侵染性克隆有广泛的研究和应用价值。     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.     

Occurrence of a new recombinant begomovirus species infecting tomato in the Al‐Batinah region of Oman

Whitefly‐transmitted begomoviruses are the most important limiting factor for tomato cultivation in Oman, particularly in the Al‐Batinah region, the major agricultural area of the country. Commercial farms in the Al‐Batinah region were surveyed during January–March 2013. Samples of tomato showing leaf curl disease symptoms typical of begomoviruses were collected and analysed. Full‐length sequences of five clones were shown to have relatively low percentage identity values to known begomoviruses, with the highest (88·6%) to isolates of Tomato leaf curl Oman virus (ToLCOMV), a begomovirus previously reported in Oman, indicating that these represent a newly identified species, for which the name Tomato leaf curl Barka virus (ToLCBrV) is proposed. Four isolates of ToLCBrV were found associated with Tomato leaf curl betasatellite (ToLCB). The five isolates of ToLCBrV characterized in this study were shown to be recombinants, with ToLCOMV as the major parent, and a fragment of Croton yellow vein virus (CrYVV) spanning the 3′ half of the replication‐associated protein. The significance of these findings is discussed.     

No detection of seed transmission of citrus tatter leaf virus in ‘Meyer’ lemon

Journal of Plant Diseases and Protection - Citrus tatter leaf virus (CTLV), a strain of apple stem grooving virus, is a virus of citrus that is of commercial importance for trifoliate orange...     

Effects of plant age on disease development and virulence of Clavibacter michiganensis subsp. michiganensis on tomato

The effect of plant age at the time of inoculation on the severity of bacterial wilt and canker disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) was examined in six greenhouse experiments. The period during which inoculations led to wilt and death of tomato plants was defined. This period, designated ‘window of vulnerability’, ranged from transplanting to the 17‐ to 18‐leaf stage. Plants inoculated after this period expressed disease symptoms but did not wilt or die. No significant changes in disease incidence were observed when leaves of different ages were inoculated. Yield accumulation was significantly reduced in plants inoculated within the window of vulnerability compared with those inoculated after this period. Expression of virulence genes, viz. celA, encoding a secreted cellulase, and the serine protease‐encoding pat‐1, chpC and ppaA, was induced during the early stages after inoculation in plants inoculated within the window of vulnerability. Differences in Cmm population between plants inoculated within and outside of this period were insignificant after the first week post‐inoculation, indicating that differences in disease severity, yield loss and expression of virulence determinants are not correlated with Cmm population level. Results suggest that implementation of precautionary measures during the window of vulnerability to avoid secondary spread of Cmm will have a season‐long effect on plant mortality and may minimize, or even prevent, yield losses.     

Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

A Variant of Cucumber mosaic virus Is Restricted to Local Lesions in Inoculated Tobacco Leaves with a Hypersensitive Response

A variant of Cucumber mosaic virus, CMV(Y/GM2), was isolated from a tobacco plant with mild green mosaic symptoms that was regenerated in vitro from a yellow strain of CMV [CMV(Y)]-infected tobacco leaves by tissue culture. CMV(Y/GM2) has two amino acid substitutions at 36 and 111 positions in the coat protein encoded on RNA3. CMV, assembled by mixing in vitro transcribed CMV(Y) RNA1 and RNA2 plus infectious RNA3 transcribed in vitro from cDNA to RNA3 of CMV(Y/GM2), was prepared and designated as CMV(Y/GM2)tr. When tobacco (Nicotiana tabacum cv. Xanthi nc) plants were inoculated with CMV(Y/GM2)tr, large necrotic local lesions in which the virus was localized, developed on the inoculated leaves. This host response unique to CMV(Y/GM2)tr was similar to the hypersensitive response (HR), which is a common resistance response to avirulent pathogens and was observed in five cultivars of Nicotiana tabacum and eight Nicotiana species. The revertant virus, however, accumulated to quite different levels in the various hosts. CMV(Y/GM2)tr induced pathogenesis-related 1 (PR-1) protein accumulation and systemic acquired resistance (SAR) which were generally observed in the HR. However, when tobaccos were inoculated with CMV(S36P)tr and CMV(V111I)tr, which have an amino acid substitution at either the 36 or 111 position in the coat protein of CMV(Y), respectively, CMV(S36P)tr was restricted to the primary infection site without necrotic local lesion formation and PR-1 protein and SAR induction. CMV(V111I)tr, however, systemically spread and induced mild green mosaic symptoms, while the host had the HR to CMV(Y/GM2)tr. The localization of CMV(Y/GM2)tr at the primary infection site may not only be caused by the HR, but also by the restriction of virus systemic movement resulting from the amino acid substitution at position 36 in the coat protein of CMV(Y). Received 15 December 1999/ Accepted in revised form 18 April 2000     

Simultaneous monitoring and quantification of Melampsora allii‐populina and Melampsora larici‐populina on infected poplar leaves using a duplex real‐time PCR assay

The rust fungi Melampsora larici‐populina (Mlp) and Melampsora allii‐populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co‐infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real‐time PCR assay targeting each species was developed, based on single‐copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time‐course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co‐inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp.