Distribution and diversity of begomoviruses in tomato and sweet pepper plants in Costa Rica

Begomoviruses (genus Begomovirus, family Geminiviridae) have emerged as important plant pathogens in tropical and subtropical regions worldwide. Although these viruses were reported during the 1970s in Costa Rica, they are still poorly known. Therefore, the objective of this study was to analyse the diversity and distribution of begomoviruses in commercial tomato and sweet pepper fields from different agricultural production systems of the major growing regions of Costa Rica. A total of 651 plants were randomly sampled from greenhouses and open field crops during 2011 and 2012 in three different geographical locations. The bipartite begomoviruses Tomato yellow mottle virus, Tomato leaf curl Sinaloa virus and Pepper golden mosaic virus, and the monopartite begomovirus Tomato yellow leaf curl virus were detected in the collected samples. The complete genome of isolates from each species was cloned and sequenced. The frequency of detection of these four begomoviruses in the analysed samples ranged from 0 to 9%, the presence, and the prevalent virus varied largely according to the geographical location, the host (tomato and pepper), and the production system (greenhouses or open fields). An association between geographical region and begomovirus species was observed suggesting that in Costa Rica the heterogeneity on climate, topography and agricultural system might influence the distribution of begomovirus species in the country. A broader survey needs to be conducted to confirm it, although these preliminary results may contribute to the management of begomoviruses in Costa Rica.     

用DDRT-PCR方法克隆小鼠精子发生早期相关基因的EST

为了避免差异显示技术中的放射性污染 ,并用之于筛选、克隆精子发生早期的相关基因 ,分离纯化了小鼠的原始精原细胞及B型精原细胞 ,提取其总RNA ,逆转录获得cDNA ,以荧光差异显示方法筛选差异表达基因。利用斑点杂交技术对差异片段进行快速鉴定以排除假阳性。选取 16条差异显著的片段做克隆测序 ,通过Gen Bank/Blast比较 ,有 7个片段属于新的EST ,且均表现为在B型精原细胞中表达强度高于原始精原细胞。提交Gen Bank获得注册号。从中选取较有意义的 3条基因片段通过半定量RT PCR方法进一步验证其表达特征。和传统的差异显示方法比较 ,文中所采用的mRNA差异显示技术可快速排除假阳性结果 ,避免同位素标记带来的放射性污染。结果表明所获得的 7个新EST均表现为B型精原细胞中高表达 ,这些表达升高的基因可能与其后精子发生过程中的一系列特殊现象 (如减数分裂、变态成形 )有关 ,为生精细胞的分化做物质上的准备。     

Characterisation and genetic diversity of pepper leafroll virus,a new bipartite begomovirus infecting pepper,bean and tomato in Peru

The complete genome of a novel bipartite begomovirus (genus Begomovirus, family Geminiviridae) was cloned from a severely diseased yellow Peruvian chili pepper (Capsicum baccatum cv. Pendulum) plant collected in the department of La Libertad, Northern Peru and full‐length sequenced. The two genomic components share a common region of 156 nucleotides with a 100% sequence identity. Analysis of the genome organisation and phylogenetic comparisons revealed that the virus is a typical New World begomovirus. The closest related begomovirus, an isolate of Tomato yellow vein streak virus (ToYVSV), shared only 76.8% nucleotide sequence identity for the DNA‐A component. Therefore, following species demarcation criteria of the International Committee on Taxonomy of Viruses, this virus isolate belongs to a new begomovirus species for which the name pepper leafroll virus (PepLRV) is proposed. Pepper plants infected with the cloned PepLRV isolate developed leaf roll symptoms similar to those observed in field‐infected plants suggesting this virus as the causal agent of the disease syndrome observed in the field. Widespread occurrence of PepLRV throughout Peru was demonstrated, infecting plants of diverse cultivated species such as tomato, pepper, common and pallar beans, and of the weed species Nicandra physaloides. Low genetic diversity was observed among PepLRV isolates present in this country with no evident geographical or temporal structure of the population, typical of a recent founder effect. This is the first report of a begomovirus infecting pepper and bean crops in Peru.     

Occurrence and molecular detection of bipartite begomovirus on tomato in Southern India

Abstract

Begomoviruses are major constraint in tomato production across the world. In India, tomato production is affected by both monopartite and bipartite begomoviruses. Monopartite were reported to infect tomato crop across India. However, bipartite were reported to infect tomato in Nothern India. In the current study, seventy begomovirus symptomatic tomato samples were collected from southernstates of India and analysed using PCR-based specific primer. The analysis revealed that 45 of them were monopartite, 23 of them were bipartite and 8 bipartite also showed the presence of betasatellite. This is the first report of bipartite begomovirus infecting tomato in Southern India.     

Genetic diversity and population structure of Pepino mosaic virus in tomato crops of Spain and Morocco

Pepino mosaic virus (PepMV, genus Potexvirus) is an emergent and highly infectious pathogen responsible for economically important diseases in tomato crops. An extensive survey of tomato plants showing PepMV‐like symptoms was carried out in 2017 to study the PepMV genetic diversity and populations structure in different tomato‐producing areas of Spain and Morocco. Molecular dot‐blot hybridization analysis showed that virus populations from Spain and Morocco were mainly composed of isolates belonging to the Chilean 2 (CH2) strain, although isolates of the European (EU) strain were detected in significant proportions in Spanish populations, mainly in mixed infections. A few isolates of the American (US1) strain were also detected in Tenerife (Canary Islands, Spain) crops. Eighty‐five isolates were randomly selected and sequenced in the genomic region that encodes the triple gene block and capsid protein genes. Our phylogenetic and population genetics analyses confirmed the presence of the CH2, EU and US1 PepMV strains. Despite the high genetic similarity observed within populations, variants were maintained at low frequency under purifying selection, and differentiation among more geographically distant locations was identified, with potential gene flow contributing to the shaping of the PepMV populations structure.     

Molecular Characterization of a Distinct Begomovirus Infecting Crassocephalum crepidioides in China

A Begomovirus isolate JH1 was obtained from Crassocephalum crepidioides showing yellow vein symptoms in Jinghong of Yunnan province, China. The complete sequence of Begomovirus DNA‐A‐like molecule of JH1 was determined to be 2745 nucleotides long. When compared with other begomoviruses, JH1 DNA‐A has the highest nucleotide sequence identity (86.4%) with that of Tobacco curly shoot virus. Comparisons of individual encoded proteins with other begomoviruses show that different parts of JH1 DNA‐A have different ancestors. The molecular data show that JH1 is a distinct Begomovirus species, for which the name Crassocephalum yellow vein virus is proposed.     

Molecular Characterization of a Distinct Begomovirus Infecting Euphorbia pulcherrima in China

Virus isolate G35 was obtained from Euphorbia pulcherrima showing leaf curl and vein thickening symptoms in Tianyang, Guangxi Province, China. The virus was transmitted by whiteflies to Nicotiana tabacum, Lycopersicon esculentum, Datura stramonium and E. pulcherrima. DNA‐A contains 2746 nucleotides, with two open reading frames (ORFs) in the virion‐sense DNA and four ORFs in the complementary‐sense DNA. When compared with the DNA‐A sequence of other begomoviruses, the total DNA‐A of isolate G35 was most closely related to that of Ageratum enation virus (79.9% sequence identity). However, the deduced coat protein of G35 is most like that of Pepper leaf curl virus from Bangladesh (94.9% amino acid sequence identity), and the AC1 of G35 is most like that of Cotton leaf curl Multan virus‐Okra (87.2% amino acid sequence identity). The molecular data showed that G35 is a distinct Begomovirus species, for which the name Euphorbia leaf curl virus (ELCV) is proposed.     

Consumer attitudes toward food crops developed by CRISPR/Cas9 in Costa Rica

Plant Cell, Tissue and Organ Culture (PCTOC) - In the original publication, only affiliation number 2 was indicated as the last author’s affiliation. In fact, the author works for both...     

Population structure and genetic diversity in four tropical tree species in Costa Rica

Despite intensified interest in conservation of tropical forests, knowledge of the population genetics of tropical forest trees remains limited. We used random amplified polymorphic DNA (RAPD) data to evaluate trends in genetic diversity and differentiation for four tropical tree species, Alchornea latifolia, Dendropanax arboreus, Inga thibaudiana and Protium glabrum . These species occur at contrasting population densities along an elevational gradient and we use RAPD and ecological data to examine natural levels of genetic diversity of each species, trends in genetic variability with population density and structure, genetic differentiation along the elevation gradient, and the relationship between genetic diversity and such factors as seed dispersal and pollination syndrome. At the distances we examined (plot distances ranging from 0.8 to 8.6 km) there was very little genetic structuring at any distance along the gradient. All four species exhibited levels of variation expected for spatial distribution, mating system and pollinator syndrome; greater than 96% of the genetic variation occurred within plots for Inga thibaudiana, Protium glabrum and Dendropanax arboreus. Alchornea latifolia only occurred in a single plot. The results of this study contribute to a growing database of genetic diversity data that can be utilized to make predictions about the effect of disturbance and subsequent reductions in population size on genetic variation and structure in tropical tree species.     

Beta diversity and oligarchic dominance in the tropical forests of Southern Costa Rica

Recent studies have reported a consistent pattern of strong dominance of a small subset of tree species in neotropical forests. These species have been called “hyperdominant” at large geographical scales and “oligarchs” at regional‐landscape scales when being abundant and frequent. Forest community assembly is shaped by environmental factors and stochastic processes, but so far the contribution of oligarchic species to the variation of community composition (i.e., beta diversity) remains poorly known. To that end, we established 20.1‐ha plots, that is, five sites with four forest types (ridge, slope and ravine primary forest, and secondary forest) per site, in humid lowland tropical forests of southwestern Costa Rica to (a) investigate how community composition responds to differences in topography, successional stage, and distance among plots for different groups of species (all, oligarch, common and rare/very rare species) and (b) identify oligarch species characterizing changes in community composition among forest types. From a total of 485 species of trees, lianas and palms recorded in this study only 27 species (i.e., 6%) were nominated as oligarch species. Oligarch species accounted for 37% of all recorded individuals and were present in at least half of the plots. Plant community composition significantly differed among forest types, thus contributing to beta diversity at the landscape scale. Oligarch species was the component best explained by geographical and topographic variables, allowing a confident characterization of the beta diversity among tropical lowland forest stands. Abstract in Spanish is available with online material.     

Genetic diversity analysis of the endangered slipper orchid Phragmipedium longifolium in Costa Rica

Phragmipedium longifolium is an endangered terrestrial orchid. In Costa Rica, these plants are found growing in small and isolated patches, some of them consisting of just four to six individuals. Information about the genetic variability within and among populations is very important for the conservation of this endangered species. A total of 160 samples were collected in six locations and analyzed with amplified fragment length polymorphism technique. The genetic diversity of P. longifolium in Costa Rica (Hw = 0.1711) is high and differentiation among sampled locations is moderate (φ_pt = 0.2013) in comparison with results of studies in some other terrestrial orchid species using the same technique. The percentage of polymorphic loci was, on average, 51.5. The analysis of molecular variance (AMOVA) indicated the main genetic variation was within sampled locations (80%), even though the variation among locations was also significant. In situ conservation is recommended because, in addition to protecting habitat and avoiding fragmentation, mycorrhizal fungi and pollinators are also protected. Close proximity between populations is required to maintain high genetic variability through a gene flow continuum. It is suggested that conservation of patches with higher genetic variability be prioritized as many are located in unprotected areas. A germplasm bank for ex situ conservation has been established in the living collection at Lankester Botanical Garden using the plants collected in this study. Finally, a search for new locations of this species is also suggested.     

Advances in diagnosing tomato yellow leaf curl geminivirus infection

As a result of the spread of TYLCV on tomato crops, reliable and rapid diagnostic tools to identify and isolate new sources of infection are necessary. We tested several methods, based both on antibodies and on nonradioactive DNA probes. Indirect plate-trapping ELISA was only effective in detecting the virus in purified preparations, but not in crude extracts. Dot-ELISA with chemiluminescence detection gave satisfactory results when young stems were directly squashed on membranes. A digoxigenin-labeled probe, detected with chemiluminescence, was used in leaf squashes and dot blots. Best results were obtained with dot blots of total nucleic acids prepared with a fast and safe procedure. TYLCV DNA was readily and reliably detected in spots corresponding to 15 μg fresh weight. When weak signals were observed, total extracts were analyzed by Southern blotting, to confirm the presence of viral DNA forms.     

对TMV不同抗性番茄品种叶绿体DNA限制性差异片段的克隆

对ＴＭＶ不同抗性番茄品种ｃｔ－ＤＮＡ经限制性内酶切后,将所获得的差异片段Ｂａｍ６克隆到质粒ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ中,经筛选、菌落原位杂交、斑点杂交及电泳鉴定,证明重组体为抗性品种Ｂａｍ６差异片段克隆,对探讨番茄ｃｔ－ＤＮＡ与抗ＴＭＶ的关系有重要意义。     

Assessment of Tuta absoluta yield loss in Iranian tomato crops

The South American tomato pinworm, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae), a devastating pest of tomato plants, originates from South America but it has been considered an invasive pest for Iran since 2010. To assess crop loss caused by this pest, a study was conducted in a 600 m² field under a randomised complete block design. The impact of different initial densities of T. absoluta, including control (without infestation), one, two, four, and eight T. absoluta eggs on each host plant in the first and second year was investigated with an additional treatment (16 pinworm eggs per host plant) used in the second year. Yield attributes including length and width of fruits, numbers of infested and intact fruits along with their weights were sampled and assessed on a weekly basis. In addition, the correlation between the number of galleries and these characteristics was also investigated. Crop loss data demonstrated that the effect of different infestation levels on fruit size and the weight of intact fruits was not significant over both years. However, the effect of treatment on the percentage and weight of infested fruits was shown to be significantly different in 2014 and 2015. Furthermore, there was a negative significant correlation between the number of galleries and weight of infested fruits in both years. Collectively, these results will be useful for enhancing pest management programs of T. absoluta in Iran.     

Genetic diversity and geographic distribution of Bemisia tabaci and Trialeurodes vaporariorum in Costa Rica

The tobacco whitefly Bemisia tabaci (Gennadius) cryptic species complex and of the greenhouse whitefly Trialeurodes vaporariorum (Westwood) are extensively reported as destructive pests in vegetable crops worldwide. A survey was conducted in 2011 and 2012 to determine the occurrence and genetic diversity present in the populations of these whiteflies in the major vegetable production areas of Costa Rica. Insect samples were collected from sweet pepper (Capsicum annuum L.), tomato (Solanum lycopersicum L.), common bean (Phaseolus vulgaris L.) and weeds present in commercial crops either in open field or greenhouse conditions. PCR‐RFLP analysis of mitochondrial cytochrome c oxidase subunit 1 gene (mtCOI) sequences of 621 whitefly individuals confirmed the presence of the Mediterranean (MED) type of the B. tabaci and of T. vaporariorum in most sampled regions. Also, individuals of the Middle East‐Asia Minor 1 (MEAM1) type of the B. tabaci were observed in low numbers. Contingency analyses based on type of crop, geographical region, whitefly species, year of collection and production system confirmed that T. vaporariorum was the most frequent species in vegetable production areas in Costa Rica, both in greenhouses and in open fields. B. tabaci MED is likely spreading to new areas of the country, whereas B. tabaci MEAM1 was mostly absent or rarely found. Comparisons of mtCOI sequences from B. tabaci individuals revealed the presence of four B. tabaci sequence haplotypes (named MED‐i, MED‐ii, MEAM1‐i, MEAM1‐xviii) in Costa Rica, three of them identical to B. tabaci haplotypes previously reported in the Western Hemisphere and other parts of the world. Analysis of sequences of T. vaporariorum individuals revealed a more complex population with the presence of 11 haplotypes, two of which were identical to T. vaporariorum sequences reported from other countries.     

Detection and elimination of Begomovirus infecting Trichosanthes dioica (pointed gourd) plants in Uttar Pradesh,India

Begomovirus is widely spread on cultivated crops in India. In a survey, infected plants of Trichosanthes dioica (Pointed gourd) with the symptoms of mosaic and leaf curling were collected and checked for Begomovirus infection through PCR analysis. Application of meristem culture technique for regeneration of virus free plants of T. dioica was assessed. Plantlets were regenerated from infected plants of T. dioica through meristem culture. Regenerated plants were found Begomovirus free as evident by PCR analysis suggesting the suitability of meristem culture technique for elimination of Begomovirus from infected plants of T. dioica. This is the first report on elimination of Begomovirus in T. dioica in India.     

Virus-induced gene silencing in tomato

We have previously demonstrated that a tobacco rattle virus (TRV)-based vector can be used in virus-induced gene silencing (VIGS) to study gene function in Nicotiana benthamiana. Here we show that recombinant TRV infects tomato plants and induces efficient gene silencing. Using this system, we suppressed the PDS, CTR1 and CTR2 genes in tomato. Suppression of CTR1 led to a constitutive ethylene response phenotype and up-regulation of an ethylene response gene, CHITINASE B. This phenotype is similar to Arabidopsis ctr1 mutant plants. We have constructed a modified TRV vector based on the GATEWAY recombination system, allowing restriction- and ligation-free cloning. Our results show that tomato expressed sequence tags (ESTs) can easily be cloned into this modified vector using a single set of primers. Using this vector, we have silenced RbcS and an endogenous gene homologous to the tomato EST cLED3L14. In the future, this modified vector system will facilitate large-scale functional analysis of tomato ESTs.     

不同邻作作物对番茄田间烟粉虱种群动态的影响

为明确不同邻作模式下番茄田间烟粉虱的种群动态与空间分布格局,于云南省昆明市选取与西葫芦、黄瓜、玫瑰等作物邻作的番茄田及单作番茄田为研究样地,通过五点取样法进行调查,采用四分位法对不同作物邻作的番茄田间烟粉虱种群动态进行分析.结果表明:在烟粉虱的主要发生期,番茄单作田间烟粉虱种群数量最高,雌成虫达26.69头?叶-1,雄...