Sequence Diversity of the Nucleoprotein Gene of Peanut bud necrosis virus Isolates from South India

Peanut bud necrosis virus (PBNV), genus Tospovirus (family Bunyaviridae), is an important virus infecting peanut and other crops in South India. PBNV isolates naturally infecting groundnut, brinjal, tomato, black gram, field bean, cowpea, cotton, jute, taro and Calotropis plants were collected from different regions of South India and characterized. Infection was confirmed by direct antigen‐coating enzyme‐linked immunosorbent assay (DAC‐ELISA) using PBNV‐specific antiserum. The coat protein gene was further amplified using PBNV coat protein‐specific primers. The amplicon (830 bp) was cloned and sequenced; sequence analysis revealed that the N gene shared 93–100% and 95–100% sequence identity with PBNV at the nucleotide and amino acid levels, respectively.     

Adh 1 first intron polymorphisms in Argentinean populations of Ceratitis capitata

DNA size polymorphisms were utilized in a study of 24 natural populations of Ceratitis capitata Wiedemann (Diptera: Tephritidae) from Argentina. The first intron of alcohol dehydrogenase 1 gene (Adh₁) was amplified using exon priming intron crossing‐polymerase chain reaction. Three size variants were detected among the 307 samples analyzed. To better differentiate the size variants, further digestion of PCR products with the EcoRI restriction enzyme was carried out. Complete nucleotide sequences of the three‐allele variants were obtained and single changes, insertions, deletions, and EcoRI recognition sites were located. Population allele frequencies were analyzed and a global mean heterozygosity (He) of 0.33 was obtained. In most populations, observed allelic frequencies conformed to Hardy–Weinberg expectations. Significant differences between provinces and sampling sites within these provinces, and among some populations were found. The average number of insects exchanged among populations (Nm) was estimated and high values were observed between Argentina and populations from two African countries (Morocco and Kenya), Australia, and Hawaii (Kauai). Pest introduction sources and dispersion patterns in Argentina are discussed based on these results as well as on available bibliographical data.     

Relationships between Tospovirus Incidence and Thrips Populations on Tomato in Mendoza, Argentina

The objectives of this work were to estimate the capability of local populations of thrips as vectors of groundnut ringspot virus (GRSV) and tomato spotted wilt virus (TSWV), and to determine the species composition of vectors in tomato crops. Transmission assays were performed. Incidence of tospoviruses was estimated in commercial crops. Random samples of flowers were taken from tomato for identification of thrips. Of the five species of thrips tested, Frankliniella gemina (first record), F. occidentalis and F. schultzei transmitted GRSV and TSWV. F. schultzei was a significantly more efficient vector of GRSV than F. occidentalis under controlled assay conditions. The thrips were identified on flowers from six surveyed tomato crops. F. occidentalis was the most frequently identified species (43.0%), followed by F. schultzei (35.6%) and Thrips tabaci (10.1%). The incidence of tospoviruses was low (1.1–2.8%) in crops planted during August–September and greater (9.5–61.1%) in crops planted in December. GRSV was prevalent (85%) over TSWV (11%).     

Molecular Detection and Complete Genome Sequences of Tomato chlorosis virus Isolates from Infectious Outbreaks in China

Tomato chlorosis virus (ToCV) is a whitefly‐transmitted, phloem‐limited, bipartite Crinivirus. In 2012, severe interveinal symptoms characteristic of ToCV infections were observed in greenhouse tomato plants in the Shandong province of China. High levels of infestation by whiteflies (Bemisia tabaci), which transmit ToCV, were also observed on tomato plants in all the greenhouses investigated. The presence of ToCV was confirmed by specific RT‐PCR either in the sampled plants or in the whiteflies collected from the ventral surface of the leaves of diseased plants. The complete genomic nucleotide sequences (RNA1 and RNA2) of the Shandong isolate of ToCV (ToCV‐SDSG) were determined and analysed. ToCV‐SDSG RNA1 consisted of 8594 nucleotides encompassing four open reading frames (ORFs). ToCV‐SDSG RNA2 consisted of 8242 nucleotides encompassing nine ORFs. Phylogenetic analysis suggests that the Chinese ToCV‐SDSG isolate is most similar to the ToCV‐Florida isolate.     

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

Genetic Characterization and Pathogenicity of Rhizoctonia solani Associated with Common Bean Web Blight in the Main Bean Growing area of Argentina

Common bean web blight (WB), caused by the fungus Rhizoctonia solani (teleomorph Thanatephorus cucumeris), is among the endemic fungal diseases of major impact in north‐western Argentina (NWA). This study aimed to analyse the genetic and pathogenic diversity of R. solani in Salta, NWA, where 97 isolates were recovered from commercial bean cultivars and wild beans showing WB symptoms in a major bean production area. The isolates were characterized on the basis of specific primers, rDNA‐ITS sequences and morphological characteristics. All the isolates were identified as R. solani AG 2‐2WB, and they exhibited considerable intragroup variation. The phylogenetic tree generated with the ITS sequences confirmed the isolates identification. Aggressiveness of the isolates towards bean seedlings was assessed in the greenhouse. A great variability in virulence was observed among the isolates analysed. On the basis of the disease reaction on foliar tissues, the isolates were grouped into three virulence categories as follows: weakly virulent (30%), moderately virulent (38%) and highly virulent (32%). However, no correlation between virulence and geographical origin was detected. The information generated in this study provides initial data on the population variability of the WB pathogen in north‐western Argentina and represents a valuable contribution to regional breeding programmes aimed to obtain cultivars with durable resistance.     

Molecular characterisation and relative incidence of bean‐ and soybean‐infecting begomoviruses in northwestern Argentina

Recent studies identified three begomoviruses infecting soybean and bean crops in northwestern (NW) Argentina, bean golden mosaic virus (BGMV), a virus with high capsid protein identity with Sida mottle virus (SiMoV) and a possible new viral species (isolate A). Analysis of complete DNA‐A sequences confirmed that isolate A represents a new viral species for which the authors propose the name soybean blistering mosaic virus (SbBMV), whereas the SiMoV‐like virus is actually an isolate of tomato yellow spot virus (ToYSV). Molecular hybridisation‐based detection of the three begomoviruses was accomplished using a general probe obtained by mixing full‐length DNA‐A clones of the three begomoviruses and specific probes comprising part of the common region of each viral genome. These probes were used to test samples collected in NW Argentina from 2004 through 2007. Fifty‐three percent of the bean samples were infected with BGMV, 11.5% with ToYSV and 9% with SbBMV. For soybean samples, 33% were infected with SbBMV and 18% with ToYSV. BGMV was not detected in soybean. ToYSV was also detected in the wild species Abutilon theophrasti.     

Nuclear insertions and heteroplasmy of mitochondrial DNA as two sources of intra‐individual genomic variation in grasshoppers

We examined the level of intra‐individual variation in a region of the mitochondrial genome coding for cytochrome oxydase 1 (COI) in two grasshopper species using a clone‐and‐sequence analysis of hundreds of sequences. In both Locusta migratoria and Chortoicetes terminifera, we found that 60–65% of the clones were unique COI‐like sequences. Among these COI‐like sequences, 70–75% diverged by less than 1% from the real mitochondrial haplotypes, and were likely to represent microheteroplasmic molecules. About 20% of the COI‐like sequences diverged by more than 9% from the mitochondrial haplotypes, and generally included stop codons, suggesting that these sequences were nuclear mitochondrial pseudogenes (NUMTs). Only six sequences, diverging by 2–6% from the mitochondrial haplotypes, were identified as potentially misleading in phylogenetic studies. In addition, we found that five sequences from C. terminifera were associated with mobile elements or repetitive DNA families.     

Identification of a distinct strain of Cotton leaf curl Gezira virus infecting tomato in Oman

Tomato (Solanum lycopersicum) plants exhibiting yellowing, curling and stunting symptoms were identified in fields of the Tawoos Agricultural Systems, in Al‐Batinah in Oman. Cloning and sequencing of restriction endonuclease digested rolling circle amplified viral DNA identified a cotton begomovirus (family Geminiviridae) associated with the symptomatic tomato plants. Detailed analysis of complete sequences showed the virus to be a previously unknown strain of Cotton leaf curl Gezira virus (CLCuGeV) in association with the betasatellite Tomato leaf curl betasatellite (ToLCB). The new CLCuGeV strain, for which the name “Al Batinah” strain is suggested, has the greatest levels of sequence identity (91.9%) to an isolate of CLCuGeV recently reported from the neighbouring United Arab Emirates. Additionally, CLCuGeV‐Al Batinah was shown to have a recombinant origin with sequences donated by an African cassava mosaic virus‐like parent. This is the first identification of this Malvaceae‐adapted begomovirus in tomato. Although ToLCB is common in Oman, being one of only two betasatellites identified there so far, this is the first identification of this betasatellite with CLCuGeV. The significance of these findings is discussed.     

Biological and Molecular Characterization of Alfalfa Mosaic Virus Infecting Trumpet Creeper (Campsis radicans) in Iran

Samples of trumpet creeper (Campsis radicans) leaves showing mottling and mosaic were collected from plants growing in a private garden in Tehran province, Iran, in 2012. Symptomatic leaf samples were tested for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) infection in enzyme‐linked immunosorbent assay (ELISA), using specific antibodies. None of the samples were positive for CMV and PSV; however, all reacted positively with that of AMV antiserum. In biological assay, systemic infection was found on Datura stramonium, Nicotiana tabacum cvs., White Burley, and Xanthi, 21 days postinoculation (DPI), while necrotic local lesions were obtained following inoculation of Phaseolus vulgaris and Vigna unguiculata within three to four DPI. Using a pair of primers specific for AMV, a DNA fragment of 880 bp was RT‐PCR‐amplified. Analysis of the sequences revealed the presence of 657 nucleotides of AMV complete coat protein (CP) gene (translating 218 amino acid residues). Phylogenetic analysis using neighbour‐joining (NJ) method clustered AMV isolates into two main types and the IRN‐Tru (GenBank Accession No. JX865593 ) isolate fell into type I. Pairwise nucleotide distances also confirmed two main types with the highest and lowest similarities for type I and II, respectively. The association of AMV with mosaic disease of C. radicans represents the first record from the world.     

Molecular Characterization of 16S Ribosomal DNA and Phylogenetic Analysis of Two X-disease Group Phytoplasmas Affecting China-tree (Melia azedarach L.) and Garlic (Allium sativum L.) in Argentina

Two phytoplasmas closely related to the X‐disease group were associated with China‐tree (Melia azedarach L.) and garlic (Allium sativum L.) decline diseases in Argentina. The present work was aimed at studying their phylogenetic relationship based on molecular characterization of the 16S ribosomal DNA sequences. Phytoplasma DNAs were obtained from naturally infected China‐tree and garlic plants from different geographical isolates. The results from analysis of restriction fragment length polymorphisms and nucleotide sequences of the 16S rDNA showed the affiliation of China‐tree and garlic decline phytoplasmas to the 16SrIII (X‐disease group), subgroups B and J, respectively. Both organisms had high sequence similarities in the 16SrDNA nucleotide sequence with the Chayote witches’ broom phytoplasma from Brazil. The phylogenetic tree, constructed by parsimony analysis, grouped the Garlic decline, China‐tree decline, Chayote witches’ broom and Clover yellow edge phytoplasmas into a cluster separated from the other phytoplasmas of the X‐disease group.     

Comparative cytopathology of Crinivirus infections in different plant hosts

We used transmission electron microscopy to compare the cytopathology induced in plants by five criniviruses (genus Crinivirus; Lettuce infectious yellows virus (LIYV), Cucurbit yellow stunting disorder virus (CYSDV), Tomato infectious chlorosis virus (TICV), Tomato chlorosis virus (ToCV) and Beet pseudo‐yellows virus (BPYV) (Hartono et al., 2003)). We also compared the patterns of infection for plants and mesophyll protoplasts infected by LIYV and Beet yellows virus (BYV), type members of genera Crinivirus and Closterovirus, respectively. The main cytopathological effects induced in plants by criniviruses were common in young leaves and included alterations of the chloroplasts and the presence of BYV‐type inclusion bodies in companion cells. Virus‐like particles were present in sieve tubes and vascular parenchyma cells as scattered particles, or in companion cells as large masses forming cross‐banded inclusions. Depending on the virus and the plant, it was possible to find virions or virus‐like particles out of the phloem cells, but only in cells of the bundle sheath. Virion‐like particles were never found outside of the vascular tissue. Accumulation of electron‐dense material at the plasmalemma was common for criniviruses, but only LIYV infections produced characteristic conical electron‐dense plasmalemma deposits (PDs). The LIYV‐induced PDs have a crystalline‐like structure and were found at the internal side of plasmalemma.     

Effects of fire frequency on survival,growth and fecundity of Paspalum quadrifarium (Lam.) in a grassland of the Flooding Pampa (Argentina)

Abstract Fire and grazing are important factors in the regulation of the grassland structure of the Flooding Pampa (Buenos Aires Province, Argentina dominated by Paspalum quadrifarium plants (henceforth ‘pajonal’)). Winter burning of pajonal is a practice that increases P. quadrifarium growth and its nutritious value for cattle. The objective of this work was to determine the responses of different demographic variables of P. quadrifarium growing in a grassland managed with different fire frequencies. The work was carried out in a pajonal situated in San Ignacio (Ayacucho county, Buenos Aires Province, Argentina). The treatments were: high fire frequency (0.8 fires per year) and low fire frequency (0.4 fires per year). The population of P. quadrifarium was affected by fire frequency. Biomass, number of tillers, and the relative production of tillers per plant did not change with fire frequency. Plant height was negatively affected by fire frequency. Whereas no plant mortality was observed in high fire frequency, 30% of the mature plants died at the end of the experiment in low fire frequency. In both treatments, plant mortality was size‐dependent and juvenile plants were more affected than mature ones. Burning had an immediate negative effect on mature and juvenile plants in terms of fecundity, survival of tillers and basal cover. Tiller survival and fecundity were significantly lower in burned plants than in unburned ones. In the high fire frequency treatment the number of ramets and/or genets and basal cover were greater than in the low fire frequency treatment.     

Nuclear spectrin-like proteins are structural actin-binding proteins in plants

Background information. Although actin is a relevant component of the plant nucleus, only three nuclear ABPs (actin‐binding proteins) have been identified in plants to date: cofilin, profilin and nuclear myosin I. Although plants lack orthologues of the main structural nuclear ABPs in animals, such as lamins, lamin‐associated proteins and nesprins, their genome does contain sequences with spectrin repeats and N‐terminal calponin homology domains for actin binding that might be distant relatives of spectrin. We investigated here whether spectrin‐like proteins could act as structural nuclear ABPs in plants. Results. We have investigated the presence of spectrins in Allium cepa meristematic nuclei by Western blotting, confocal and electron microscopy, using antibodies against α‐ and β‐spectrin chains that cross‐react in plant nuclei. Their role as nuclear ABPs was analysed by co‐immunoprecipitation and IF (immunofluorescence) co‐localization and their association with the nuclear matrix was investigated by sequential extraction of nuclei with non‐ionic detergent, and in low‐ and high‐salt buffers after nuclease digestion. Our results demonstrate the existence of several spectrin‐like proteins in the nucleus of onion cells that have different intranuclear distributions in asynchronous meristematic populations and associate with the nuclear matrix. These nuclear proteins co‐immunoprecipitate and co‐localize with actin. Conclusions. These results reveal that the plant nucleus contains spectrin‐like proteins that are structural nuclear components and function as ABPs. Their intranuclear distribution suggests that plant nuclear spectrin‐like proteins could be involved in multiple nuclear functions.     

Functional acclimation to solar UV-B radiation in Gunnera magellanica, a native plant species of southernmost Patagonia

The ecosystems of Tierra del Fuego (in southern Patagonia, Argentina) are seasonally exposed to elevated levels of ultraviolet‐B radiation (UV‐B: 280–315 nm), due to the passage of the ‘ozone hole’ over this region. In the experiments reported in this article the effects of solar UV‐B and UV‐A (315–400 nm) on two UV‐B defence‐related processes: the accumulation of protective UV‐absorbing compounds and DNA repair, were tested. It was found that the accumulation of UV‐absorbing sunscreens in Gunnera magellanica leaves was not affected by plant exposure to ambient UV radiation. Photorepair was the predominant mechanism of cyclobutane‐pyrimidine dimer (CPD) removal in G. magellanica. Plants exposed to solar UV had higher CPD repair capacity under optimal conditions of temperature (25 °C) than plants grown under attenuated UV. There was no measurable repair at 8 °C. The rates of CPD repair in G. magellanica plants were modest in comparison with other species and, under equivalent conditions, were about 50% lower than the repair rates of Arabidopsis thaliana (Ler ecotype). Collectively our results suggest that the susceptibility of G. magellanica plants to current ambient levels of solar UV‐B in southern Patagonia may be related to a low DNA repair capacity.