Detection and Characterization of Phytoplasma and Sugarcane Yellow Leaf Virus Associated with Leaf Yellowing of Sugarcane

Leaves from sugarcane were collected from Egyptian plantation fields and tested for phytoplasma (Sugarcane yellows phytoplasma, SCYP) and Sugarcane yellow leaf virus (SCYLV) using nested PCR (with different primers) and RT‐PCR, respectively. These results showed significant differences in the amplification of the PCR assays. The primer MLO‐X/MLO‐Y, which amplified the 16S‐23S rDNA spacer region, was the most precise to detect the phytoplasma in sugarcane plants. Sequencing and restriction fragment length polymorphism analysis revealed that all tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group, with the exception of cultivar G84‐47 belonged to the 16SrXI (Rice yellow dwarf phytoplasma) group. Three Egyptian sugarcane cultivars were phytoplasma free. Phylogenetic analyses of 34 screened accessions of 16S ribosomal DNA gene sequences of Candidatus phytoplasma including the ones collected from Egypt used in this study and those extracted from GenBank showed that they split into two distinct clusters. The phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. All tested Egyptian sugarcane plants were infected by SCYLV with the exception of cultivar Phil‐8013 which was virus free.     

Molecular characterization of a new badnavirus associated with streak symptoms on enset (Ensete ventricosum,Musaceae)

Electron microscopy of leaf samples displaying streak symptoms from enset (Ensete ventricosum), a banana‐like plant widely cultivated in Ethiopia, showed the presence of bacilliform shaped virions as known for badnaviruses. DNA extracts subjected to rolling circle amplification (RCA), polymerase chain reaction (PCR) and cloning and sequence analysis revealed that the virus has a circular double‐stranded DNA genome of 7,163 nucleotides encoding predicted proteins of 21.5 kDa, 14.5 kDa and 202.5 kDa, a genome organization known for badnaviruses. The virus is phylogenetically most closely related to Sugarcane bacilliform Guadeloupe D virus with a nucleotide sequence identity of 77.2% at the conserved RT/RNase‐H region and 73.6% for the whole genome. Following the current species demarcation criteria, the virus should be considered as an isolate of a new species in the genus Badnavirus for which the name Enset leaf streak virus (ELSV) is suggested. Leaf samples from enset and banana were screened using virus‐specific primers, and ELSV was detected in six of 40 enset but not found in any of 61 banana samples. On the other hand, Banana streak OL virus (BSOLV) was detected from the majority (60%) of symptomatic banana samples but not from enset samples. This paper reports the first full‐genome sequence of a putative new badnavirus species infecting plants in the genus Ensete. In addition, this is the first report of the occurrence of BSOLV in Ethiopia.     

Molecular Diversities of Sugarcane mosaic virus and Sorghum mosaic virus Isolates from Yunnan Province,China

Degenerate Potyviridae primers were used to amplify and sequence the 3′‐terminal regions of viruses from traditional and modern cultivars of sugarcane with mosaic disease growing in different areas of Yunnan province, China. Seven samples contained Sugarcane mosaic virus (SCMV), 11 contained Sorghum mosaic virus (SrMV) and two contained both viruses. SCMV was only isolated from traditional cultivars. In a phylogenetic analysis of the partial NIb and complete coat protein coding regions, most SCMV isolates formed a distinctive phylogenetic cluster (named SO) that otherwise contained only three Vietnamese isolates. SCMV variation seems mostly related to host genotype. In the same analysis, the SrMV isolates formed three major groups, one of which is reported for the first time, but the significance of the grouping is unclear.     

Genetic diversity and population structure of Sorghum mosaic virus infecting Saccharum spp. hybrids

Sugarcane mosaic disease is widespread in many countries and has been identified to be caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV). Viral surveys of SCMV, SrMV and SCSMV were performed from 104 leaf samples of Saccharum spp. hybrid growing in China and two leaf samples in Myanmar. Sorghum mosaic virus was a major causal agent for sugarcane mosaic disease in China whereby 72.1% (75/104) of samples had SrMV infection alone, 6.7% (7/104) were mixed with SCMV and 17.3% (18/104) were mixed with SCSMV. Sugarcane streak mosaic virus infection alone occurred in 3.8% (4/104) of samples, but no single infections were observed for SCMV. Two viruses (SrMV and SCSMV) were detected in sugarcane mosaic samples in Myanmar. Phylogenetic analysis revealed that all of the SrMV isolates were clustered into three major lineages encompassing six phylogroups/genotypes based on the CP sequences (825 nucleotides) of 113 Chinese and 2 Burmese isolates from this study and 73 isolates reported worldwide. Six clearly distinct SrMV phylogroups (G1–G6) were formed and shared 74.3–94.1% nucleotide identity and 84.7–98.1% amino acid identity of CP sequences. SrMV‐G5 was identified to be new distinct phylogroup that was restricted to the Fujian and Guangxi provinces. The unique SrMV‐G6 phylogroup only occurred in Yunnan province. Insertion/deletion mutations, negative selection and frequent gene flow are factors driving the genetic evolution and population structure of SrMV in China.     

A variable region of the Sugarcane Bacilliform Virus (SCBV) genome can be used to generate promoters for transgene expression in sugarcane

Four promoters derived from sugarcane bacilliform virus (SCBV) were compared and characterised. Three were obtained by PCR amplification of purified virion DNA extracted from three sugarcane cultivars. The fourth promoter was obtained by subcloning from an almost genome-length clone of SCBV. All promoters were able to drive stable expression of -glucuronidase in sugarcane. The PCR-derived promoter sequences shared more DNA homology with banana streak virus than to the subcloned SCBV. The subcloned promoter was the strongest expressing and was able to drive reporter gene expression in vitro and in the leaves, meristems and roots of glasshouse-grown sugarcane. Expression levels were at least equal to or higher than those measured for the maize polyubiquitin promoter.     

Tissue specificity of the sugarcane bacilliform virus promoter in oat,barley and wheat

The tissue-specificity of the sugarcane bacilliform virus (SCBV) promoter was investigated in oat, barley, and wheat to determine whether its expression pattern in one species was predictive of promoter specificity in the other closely related Gramineae species. Progeny of transgenic plants produced using constructs containing the SCBV promoter driving gusA were sampled at different stages of plant development and stained for GUS activity using a histochemical assay. Overall, the GUS staining patterns were most similar between oat and barley. In all three species, similar GUS staining patterns were observed in mature endosperms, leaves, and floral bracts of developing infloresences. No GUS staining was detected in oat embryos whereas the entire barley embryo was stained, and GUS staining was confined to the scutellum of wheat embryos. Oat and barley stems exhibited GUS staining whereas no GUS staining was observed in stems of the transgenic wheat plants. The SCBV promoter conferred strong GUS staining intensity in most tissues of oat and barley but was generally weaker in wheat. These differences in SCBV promoter specificity indicate that promoter evaluation should be conducted in the target species of interest rather than by extrapolation from expression patterns in other species.     

甘蔗条纹花叶病毒P3蛋白与甘蔗Rubisco大亚基互作的研究

该研究以甘蔗条纹花叶病毒(Sugarcane streak mosaic virus,SCSMV)的P3蛋白(SCSMV-P3)为诱饵,采用酵母双杂交技术(yeast two-hybrid system,Y2HS)从甘蔗酵母文库中筛选到1个编码核酮糖-1,5-二磷酸羧化酶/加氧酶(ribulose-1,5-bisphosphate carboxylase/oxygenase,Rubisco)大亚基的基因,命名为ScRbcL。序列分析表明,该基因开放读码框(open reading frame,ORF)长度为1 337bp,编码478个氨基酸残基。Y2HS和双分子荧光互补(bimolecular flourescence complementation,BiFC)实验表明,ScRbcL与SCSMV-P3存在互作关系。研究认为SCSMV-P3与ScRbcL的互作可能是SCSMV侵染甘蔗导致花叶症状发生的分子基础。     

Molecular and Pathological Characterization of Colletotrichum falcatum Infecting Subtropical Indian Sugarcane

Red rot, caused by Colletotrichum falcatum, is the most significant problem of sugarcane worldwide. Pathological studies and three different marker systems were used to characterize 25 C. falcatum isolates collected from 18 subtropical sugarcane cultivars from 15 different sugarcane‐growing regions of three north‐eastern states of India to assess pathogen diversity. Of these 25 isolates, three were new (RR2A, RR15, RR83) from cultivars Co 7717, Co J83 and Co S88230, respectively, pathologically characterized on 13 standard differential hosts. Isolates Cf 01, Cf 08 and RR15 were the most, and Cf‐07 the least virulent. Molecular characterization using random amplified polymorphic DNA, universal rice primers (URP) and inter simple sequence repeat markers amplified a total of 161 alleles of which 159 were polymorphic (98.76%). Unweighted paired group method with arithmetic averages analysis of combined data of all the DNA markers obtained by three marker systems classified 25 isolates into six clusters at 34% genetic similarity with high Mantel matrix correlation (r = 0.83). The principal component analysis (PCA) of marker data explained 68% of the variation by first three components. Molecular diversity as revealed in these isolates is very high, but non‐structured. Isolate Co Pant 84212 was found to be genetically most diverse. We demonstrated for the first time that URPs derived from weed rice could successfully assess genetic diversity in C. falcatum. Molecular characterization of the C. falcatum isolates prevalent in north‐eastern India would enable red rot management strate‐gies, selection for resistance genes and development of resistant cultivars.     

Elimination of five viruses from sugarcane using in vitro culture of axillary buds and apical meristems

Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus elimination through meristem tissue culture originating from both apical and axillary shoots were compared. The average rates of regeneration and elongation from apical meristem tissues were 91 and 66%, respectively, with the virus-free rate among elongated shoots ranging from 61–92%. Mature axillary buds were cultivated in vitro to produce axillary shoots, from which meristem tissues were excised and cultured. These meristem tissues regenerated (77–100%) and elongated (55–88%) in culture medium at approximately the same rate as the apical meristems. The average virus elimination rate was 90% among elongated shoots derived from mature axillary buds. All five viruses can be eliminated by meristem tissue culture from both apical and axillary shoots using a standardized procedure. The overall average efficiency of virus-free plant production was 45 and 58% from apical and axillary shoots, respectively. There were no significant differences for shoot induction or virus elimination when the meristems were harvested from either the apical or the axillary shoots. This is the first report of SrMV or SCSMV elimination from sugarcane, as well as elimination of any mixed virus infections. This new method of harvesting meristems from axillary buds greatly expands the amount of material available for therapeutic treatments and thereby increases the probability of eliminating viruses from infected sugarcane.     

Molecular Characterization of the 3'-Terminal Region of Turnip mosaic virus Isolates from Eastern China

Turnip mosaic virus (TuMV; genus Potyvirus, family Potyviridae) causes great losses to cruciferous crop production worldwide. The 3′‐terminal genomic sequences of eight TuMV isolates from eastern China were compared with those of 74 other Chinese TuMV isolates of known host origin in the GenBank and isolated during the past 25 years. The reported sequences of the eight TuMV isolates are 1125 or 1126‐nucleotides (nt) long excluding the poly(A) tail. They all contain one partial open reading frame of 912 nt, encoding 304 amino acids, followed by a stop codon and a non‐translated region of 209–210 nt. Results of phylogenetic analyses showed that Chinese TuMV isolates clustered into three groups: basal‐BR, Asian‐BR and world‐B. The ratios of non‐synonymous and synonymous substitutions and results of amino acid alignment provided evidence for purifying or negative selection in TuMV populations of China.     

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit‐growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT‐PCR using designed CCYV‐specific primer pair (CCYV‐F/CCYV‐R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non‐Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.     

Elimination of virus and rapid propagation of disease-free sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. cultivar NCo376) using apical meristem culture

The use of apical meristem culture for simultaneous virus elimination and shoot proliferation in sugarcane was assessed. Virus-free plants were propagated from Sugarcane mosaic virus and Sugarcane yellow leaf virus-infected material of the South African commercial cultivar, NCo376. A combination of thermotherapy by hot water treatment of stem sections (nodes) and subsequent germination of vegetative buds at 40°C and optimal meristem size were key factors for the production of virus-free plants. Only meristems of 2 mm in length or of a smaller size (but >0.5 mm) resulted in virus-free sugarcane. Shoot induction and proliferation via direct organogenesis were achieved on Murashige and Skoog nutrient medium supplemented with 0.1 mg l⁻¹ 6-benzyladenine and 0.015 mg l⁻¹ 6-furfurylaminopurine (KIN). The established protocol provides for the rapid proliferation of virus-free shoots from infected sugarcane plants and approximately 1,300 shoots were propagated from a single 2 mm meristem in 11 weeks. Plants remained virus-free when tested 12 months later.     

Genetic transformation with untranslatable coat protein gene of <Emphasis Type="Italic">sugarcane yellow leaf virus</Emphasis> reduces virus titers in sugarcane

Sugarcane yellow leaf syndrome, characterized by a yellowing of the leaf midrib followed by leaf necrosis and growth suppression, is caused by sugarcane yellow leaf virus (SCYLV). We produced SCYLV-resistant transgenic sugarcane from a susceptible cultivar (H62-4671) and determined the amount of virus present following inoculation. The transgenic plants were produced through biolistic bombardment of cell cultures with an untranslatable coat protein gene. Presence of the transgene in regenerated plants was confirmed using PCR and Southern blot analysis. The transgenic lines were inoculated by viruliferous aphids and the level of SCYLV in the plants was determined. Six out of nine transgenic lines had at least 10³-fold lower virus titer than the non-transformed, susceptible parent line. This resistance level, as measured by virus titer and symptom development, was similar to that of a resistant cultivar (H78-4153). The selected SCYLV-resistant transgenic sugarcane lines will be available for integration of the resistance gene into other commercial cultivars and for quantification of viral effects on yield.     

Low Genetic Diversity of the Coat Protein Gene among Blueberry Red Ringspot Virus Isolates

Blueberry red ringspot virus (BRRSV) isolates have been investigated for genetic diversity. Nucleotide sequences of the coat protein (CP) gene of 19 isolates from Poland, Czech Republic, Slovenia and the United States were analysed. The nucleotide and amino acid sequence identity were 92–100% and 89–100%, respectively. Estimations of the distribution of synonymous and non‐synonymous changes indicated negative selection within the analysed CP gene and confirmed the genetic stability of the virus. At a capsid protein level, our results revealed BRRSV to be distinct from other, recombination‐prone pararetroviruses.     

Occurrence and Diversity of Pome Fruit Viruses in Apple and Pear Orchards in Latvia

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus are economically important viruses infecting fruit tree species worldwide. To evaluate the occurrence of these pome fruit viruses in Latvia, a large‐scale survey was carried out in 2007. Collected samples were tested for infection by DAS ELISA and multiplex RT‐PCR. The accuracy of the detection of the viruses in multiplex RT‐PCR was confirmed by sequencing amplified PCR fragments. The results showed a wide occurrence of viruses in apple and pear commercial orchards established from non‐tested planting material. More than 89% of the tested apple trees and more than 60% of pear trees were infected with one or more pome fruit viruses. Analyses showed that the high occurrence of viruses in several apple cultivars is due to the propagation of infected clonal rootstocks and scions from infected mother trees. Sequence analyses targeting the 3′‐terminal region of the tested viruses showed various degrees of genetic diversity within respective virus isolates. This is the first report of the occurrence of ACLSV, ASGV and ASPV in apple and pear trees in Latvia and demonstrates their genetic diversity in different host genotypes.