Apple Chlorotic Leaf Spot Virus Research Articles

First Report of Three Bunya-Like Viruses, Apple Luteovirus 1, and Apple Hammerhead Viroid in Apples from Hakkari, Türkiye

Türkiye is a major apple fruit producer in the crossroads of Europe and the Middle East. Several reports have described the presence of multiple viruses affecting apple production in Türkiye, including apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leafspot virus (ACLSV), and apple mosaic virus (ApMV) (Kurçman 1977; Fidan 1994; Çağlayan et al. 2003). However, there are no reports of the presence of the recently discovered bunya-like viruses citrus concave gum-associated virus (CCGaV), and apple rubbery wood viruses 1 and 2 (ARWV1 and 2), as well as apple luteovirus 1 (ALV-1), and apple hammerhead viroid (AHVd) in Türkiye, all of which have been previously reported in other apple-producing countries (Wright et al. 2018; Liu et al. 2018; Zhang et al. 2014). Leaves from one Gala, two Granny Smith, and one Golden Delicious apple trees showing mild symptoms of curling, chlorosis, and yellowing were collected from four different orchards in the province of Hakkari, southeast Türkiye during June 2022 and sent to USDA APHIS Plant Germplasm Quarantine Program (under permit) for virus and viroid HTS-based diagnostics. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s guidelines to prepare RNAseq libraries using the TruSeq Stranded Total RNA Library Plant Kit (Illumina, Inc) as described in Malapi-Wight et al. (2021). Libraries were sequenced on the NextSeq500 sequencer (PE 2x75), and approximately 45 million reads were obtained per each sample on average. Bioinformatic analysis was performed as described in Costa et al. (2022) using Phytopipe, where unclassified pathogen-derived reads were de novo assembled and contigs were compared to the NCBI viral nucleotide and protein databases by BlastN and BlastX respectively using a 10-4 e-value cutoff. Nearly complete genome contigs were obtained for ACLSV (OR640150) and ASPV (OR640151) in all four samples and for ASGV (OR640152) in 3 of the 4 samples. The average BlastN identity to sequences in GenBank was 92.3% for ACLSV, ranging from 89-94 %. BlastN identity for ASPV was 86%, ranging from 81-92 % while the ASGV average BlastN identity was 98.2%. Nearly complete genomes with average genome coverage of 92.4% and 95.6% for RNA1 and RNA2 of CCGaV (OR640153 and OR640154), were found in two of the four samples with BlastN identity of 94.7% and 94.8% to GenBank sequences. Additionally, nearly complete genome of the large (L), medium (M), and small (S) segments for ARWV1 were found in two samples with average genome coverage of 99.9%, 99.4%, and 100% respectively and BlastN identity of 98.8%, 95.2%, and 98.4% (OR640155, OR640156, OR640157). ARWV2 contigs were also found in 1 sample where M and S segments had a coverage of 99.8% and BlastN identity of 95.4% (OR640158 and OR640159). The nearly complete genome of ALV-1 was also found in two of four samples with genome coverage of 94.1% and an average BlastN identity of 93.4% (OR640160). AHVd was found in one of the Granny Smith trees with 19,260 mapped reads to the reference GenBank MH049335.1 and identity of 98.3% (OR640149). The HTS findings of CCGaV, ARWV1, ARWV2, and ALV-1, from Türkiye were later confirmed by Sanger sequencing using custom-designed primers targeting the coat protein, the RNA-dependent RNA polymerase, or ~390bp for the AHVd genome (Supplementary Table 1). To further learn about the incidence of these agents, we tested 12 other apple samples from six different neighboring orchards and found them at 18.8% rate for CCGaV, 12.5% for both ARWV1 and ARWV2, 25% for ALV-1, and 37.5% for AHVd respectively. To our knowledge, this is the first report of the apple viruses CCGaV, ARWV1, ARWV2, and ALV-1, and the AHVd viroid in Türkiye. Further studies of the impact of these agents on orchard’s health are necessary, including their prevalence in high apple production regions of Türkiye.

Viruses of Apple Are Seedborne but Likely Not Vertically Transmitted.

Many viruses occur in apple (Malus domestica (Borkh.)), but no information is available on their seed transmissibility. Here, we report that six viruses infecting apple trees, namely, apple chlorotic leaf spot virus (ACLSV), apple green crinkle-associated virus (AGCaV), apple rubbery wood virus 2 (ARWV2), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), and citrus concave gum-associated virus (CCGaV) occur in seeds extracted from apple fruits produced by infected maternal trees. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR) assays revealed the presence of these six viruses in untreated apple seeds with incidence rates ranging from 20% to 96%. Furthermore, ASPV was detected by RT-PCR in the flesh and peel of fruits produced by infected maternal trees, as well as from seeds extracted from apple fruits sold for fresh consumption. Finally, a large-scale seedling grow-out experiment failed to detect ACLSV, ASGV, or ASPV in over 1000 progeny derived from sodium hypochlorite surface sterilized seeds extracted from fruits produced by infected maternal trees, suggesting no detectable transmission via embryonic tissue. This is the first report on the seedborne nature of apple-infecting viruses.

An update on apple chlorotic leaf spot virus status of sweet cherry in Bulgaria

The sweet cherry (Prunus avium L.) is one of the most important stone fruit species in Bulgaria. The cherry is susceptible to many viruses. To gain a better insight into the phyto-virologic status of sweet cherry, a survey for the presence of apple chlorotic leaf spot virus (ACLSV), cherry leafroll virus (CLRVR) and raspberry ringspot virus (RpRSV), in addition to ilarviruses, was carried out in 32 commercial and 6 collection orchards in ten regions of Bulgaria between 2017 and 2022. A total of 1503 sweet cherry samples were collected from symptomatic and symptomless trees. These samples were tested by cocktail ELISA for ACLSV and DAS-ELISA for CLRVR, RpRSV ?nd additionally for prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) to detect mixed infections. Of the three viruses studied, only ACLSV was detected in commercial and collection sweet cherry orchards in all regions with intensive cherry cultivation in Bulgaria, in addition to ilarviruses. The extent of ACLSV virus infection was 8.8%. Single ACLSV infection was the most common, affecting 79.6% of infected sweet cherry trees, followed by ACLSV and PDV (14.4%) and least frequently by ACLSV and PNRSV (6.0%). The presence of ACLSV in orchards of different ages, including ?young? orchards, indicates that virus-free propagation material must be used when establishing new orchards to prevent the spread of pathogens with the planting material.

Genome analysis of viruses of Phenuiviridae, Betaflexiviridae and Bromoviridae, and apple scar skin viroid in pear by high-throughput sequencing revealing host expansion of a rubodvirus and an ilarvirus

Pear (Pyrus spp.) is an important perennial fruit crop of India. During May 2022, pear leaf samples were collected from the orchard of ICAR-Indian Agricultural Research Institute, Regional Station, Dhanda, Shimla, Himachal Pradesh, India. RNA sequencing of two pooled samples, Pe1 and Pe2, was done using NovaSeq 6000. Host unaligned reads were de novo assembled into contigs and annotated through BLAST using the virus database. Near complete genome sequences of apple rubbery wood virus 1 (ARWV-1), apple necrotic mosaic virus (ApNMV), citrus virus A (CiVA), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), and a genetic variant of ASGV named as ASGV-Shimla, and a partial sequence of apple scar skin viroid (ASSVd) were assembled. ARWV-1 and ApNMV belong to the genus Rubodvirus and Ilarvirus having tripartite negative sense and positive sense single stranded RNA genomes, respectively, and their presence in pear trees was observed for the first time indicating the host expansion. Presence of CiVA, ASGV and ACLSV in pear in India was also recorded for the first time in India. The fragments per kilobase of transcript per million reads mapped estimation showed the dominance of ASPV and CiVA in Pe1 and Pe2, respectively. Single nucleotide variants (SNVs) analysis revealed that ASPV had the highest number of SNVs (477), while ACLSV had the lowest number of SNVs (1). The presence of ARWV-1, ApNMV, CiVA, ASPV, ASGV, ACLSV, ASGV-Shimla, and ASSVd was also confirmed in pear samples through Sanger sequencing. The present study widens our understanding of the pear virome and host range expansion of viruses in the genus Rubodvirus and Ilarvirus. This information will be useful in biological study of these viruses and virus indexing programs in pear.

In Silico Apple Genome-Encoded MicroRNA Target Binding Sites Targeting Apple Chlorotic Leaf Spot Virus

Apple chlorotic leaf spot virus (ACLSV) (genus, Trichovirus; family, Betaflexiviridae) is a widespread, deleterious, and the most damaging pathogen of pome and fruit trees including domesticated apple (Malus × domestica Borkh.), to which it is transmitted by grafting and pruning. The positive-sense, single-stranded RNA virus is 600–700 nm long and has a genome of 74.7–7.56 kbp in size, minus the poly-A tail and 3′- and 5′-untranslated regions. The genome has three overlapping open reading frames (ORFs) that encode a replication-associated protein (Rep), movement protein (MP), and coat protein (CP). RNA interference (RNAi)-mediated antiviral defense in eukaryotes has evolved to control infections in plant viruses. The objective of this study was to analyze locus-derived microRNAs (mdm-miRNAs) in the apple genome with potential for targeting ACLSV +ssRNA-encoded mRNAs, using a predictive approach that involves four algorithms. The goal is to mobilize the in silico-predicted endogenous mdm-miRNAs and trigger the RNAi pathway experimentally in apple trees to evaluate antiviral resistance to ACLSV. Experimentally validated apple (2n = 2X = 34) mdm-miRNAs (n = 322) were obtained from the miRBase database and aligned to the ACLSV genome (KU870525). Of the 322 targeting mature locus-derived mdm-miRNAs analyzed, nine apple mdm-miRNA homologs (mdm-miR395k, mdm-miR5225c, and mdm-miR7121 (a, b, c, d, e, f, g, h) were predicted by all “four algorithms”, whereas fifty-eight mdm-miRNAs were identified as consensus binding sites by the combined results of two algorithms. The miRanda, RNA22, and TAPIR algorithms predicted binding of mdm-miR395k at nucleotide position 4691 and identified it as the most effective interacting mdm-miRNA targeting the virus ORF1 sequence. An integrated Circos plot was generated to validate the accuracy of target prediction and determine if apple mdm-miRNAs could bind to the predicted ACLSV mRNA target(s). A genome-wide in silico-predicted miRNA-mediated target gene regulatory network was implicated to validate interactions necessary to warrant in vivo analysis. The availability of validated locus-derived microRNAs (mdm-miRNAs) with predicted potential to target ACLSV in infected apple trees represents the first step toward development of ACLSV-resistant apple trees.

Global Spread Dynamics and Intrinsic Evolutionary Dynamics of Apple Chlorotic Leaf Spot Virus Whole-Genome Sequences over the Last 30 Years

Apple chlorotic leaf spot virus (ACLSV; genus Trichovirus) is an economically important virus. Approximately 101 ACLSV whole-genome sequences were obtained from NCBI and used to explore the evolutionary dynamics of ACLSV. The time to the most recent common ancestor of ACLSV based on BEAST analysis appeared on an apple host from Canada in 1918 and then spread around the world in three ways concurrent with host spread. The maximum clade credibility tree of ACLSV shows that the mean evolution rate was 4.92 × 10-4 substitutions per site per year (subs/site/year), and we found that, during host evolution, the rate of evolution was mostly 2.31 × 10-4 to 2.72 × 10-4 subs/site/year. The rate of geographic evolution was 5.51 × 10-4 to 6.17 × 10-4 subs/site/year. To further explore the intrinsic changes in ACLSV during the process of geographic and host spread, we explored the secondary structural changes of the ACLSV coat protein (CP), which were mainly concentrated in four regions-20 to 40, 70 to 90, 120 to 140, and 180 to 193-which are related to the presence or absence and change in length of the β-turn, β strand, coil, and α helix, respectively. We then explored the codon usage preference within the CP across the migration pathways of ACLSV. These comprehensive analyses not only reveal the changes in ACLSV in the last 30 years but also further elucidate the intrinsic evolutionary dynamics of ACLSV. This is also the first report on the intrinsic evolutionary dynamics of ACLSV.

Simultaneous detection of five viruses and two viroids affecting apples through a DNA macroarray chip

Multiple infections of various viruses and viroids in apple trees are common and have caused a significant loss in the world apple industry. To provide an early detection of any of those possible pathogens at the molecular level, a multiplex DNA macroarray chip was designed and developed for a simultaneous identification of five common apple viruses and two viroids including apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple mosaic virus (ApMV), apple necrosis mosaic virus (ApNMV), apple scar skin viroid (ASSVd), and apple dimple fruit viroid (ADFVd). The macroarray with a 23 bp probe arranged with the coat protein (CP) gene or a target DNA segment of each viruses and viroids has demonstrated a high specificity and sensitivity without any competitions, inhibitions or cross-interferences when it was tested against more than a mixture of viral and viroid samples. To our best knowledge, this is the first report on the simultaneous detection of five different apple viruses and two viroids through using a DNA macroarray, therefore, we suggest that this detection protocol and procedure be used for any apple viral diagnosis before setting up a production nursery for virus-free apple seedlings.

Genetic diversity and phylogenetic relationships of apple chlorotic leaf spot virus isolates from Malus, Pyrus and Prunus hosts in Latvia

AbstractThe prevalence of apple chlorotic leaf spot virus (ACLSV) in different Rosaceae species, mainly from cultivated apple (Malus domestica), pear (Pyrus communis), plum (Prunus domestica), apricot (Prunus armeniaca) and wild plants of genus Amelanchier, Cotoneaster, Crataegus, Malus, Prunus and Sorbus were studied. ACLSV was found in cultivated M. domestica, P. communis, P. domestica and wild‐growing Malus, including M. sylvestris. Thirty‐four sequences of the ACLSV coat protein gene were obtained and compared with 372 homologous sequences from GenBank to evaluate the genetic diversity of the virus in Latvia and worldwide. The sequence identities varied from 79.6% to 98.4% at the nucleotide level and 84.5% to 100% at the amino acid level. Phylogenetic analyses clustered ACLSV pome fruit isolates into three genetic types but isolates from stone fruits into four phylogenetic groups. Three genetic types of ACLSV pome and stone fruit isolates were identified in Latvia. Two of them were previously characterized as type B6 and P205. The third genetic type was newly recognized among the stone fruit isolates and distinguished from other virus isolates by amino acid substitutions in three positions, Ser14, Asn23 and Leu72. Two natural recombinants, LV27 and NZ5, were detected among ACLSV isolates from Latvia, and recombination events were identified for the first time in wild‐growing Malus, confirming that recombination is a common process in the ACLSV population.

Virusi i viroidi kruške

One of the most common fruit species in the world is the pears. Compared to stone and small fruit trees, pears and other pome fruits are infected by a lesser number of viruses and viroids. The majority of commercial pear cultivars do not exhibit symptoms on their leaves, fruits, or other plant parts due to viruses. Only foliage and fruits show symptoms in susceptible cultivars. Apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leafspot virus (ACLSV), and apple mosaic virus (ApMV) are widespread and economically significant pear viruses. Pear blister canker viroid (PBCVd) and apple scar skin viroid (ASSVd) are the most dangerous viroids infecting pears.

The effect of the Pharmaiod preparation in the virus elimination of pear plants using the thermotherapy method

Currently, the main methods of virus elimination of fruit crops are dry-air thermotherapy, meristem culture and chemotherapy. The use of a complex of physical and chemical methods makes it possible to increase the efficiency of virus elimination. Improving the methods of recovery must be carried out in relation to certain types of viruses, the biological characteristics of the crop and variety. The purpose of the research is to study the effect of the Pharmaiod preparation on biometric, physiological parameters and the release of harmful viruses-free pear plants in the process of dry-air thermotherapy. Improvement of pear plants of varieties Letnyaya Zabava, Zolotoy Vityaz, hybrids R-11-9, R-10-3 and R-2-4 from harmful latent viruses of Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) were carried out in 2020-2021 at the temperature of 38 °C for 3 months using a thermal chamber designed by the Federal Horticultural Research Centеr for Breeding, Agrotechnology and Nursery. Biometric parameters in pear plants depended on varietal characteristics, the duration of therapy and the concentration of the Pharmaiod preparation. In most of the varieties and forms studied, after 3 months of thermotherapy, the Pharmaiod preparation at a concentration of 1 ml/l contributed to an increase in the length of 1 shoot of pear plants by 1.4-3.1 times compared with the control. Under the conditions of a thermal chamber, according to a set of indicators, pear hybrids R-11-9 and R-10-3 were characterized by the highest heat resistance; medium heat resistance was shown by Letnyaya Zabava and Zolotoy Vityaz varieties; low – by R-2-4 hybrid. Higher heat-resistant varieties and forms were characterized by a more restrained growth of shoots in length compared to lower heat-resistant ones. An average negative significant correlation at the 5% significance level was established between the water content in the leaves and the length of 1 shoot (r = -0.52). The yield of virus-free pear plants depended on the type of virus. The yield of pear plants free from 4 major harmful viruses as a result of dry-air thermotherapy without the use of the Pharmaiod preparation was 50 %, with the use of the drug it was 60 %.

Apricot breeding for multiple resistance to Plum pox virus and Apple chlorotic leaf spot virus

Spain is one of the most important producers of fresh apricots (Prunus armeniaca L.). Traditional production was based on several local cultivars with good quality and well adapted to the industry. However, most are susceptible to important viral diseases that affect fruit production and quality. The main viral diseases are sharka (Plum pox virus, PPV) and viruela (Apple chlorotic leaf spot virus, ACLSV). Using foreign resistance sources to PPV, new resistant cultivars have been released. More recently, two new resistance sources to ACLSV have been identified: the Spanish cultivar ‘Mauricio’ and the French cultivar ‘Bergeron’. With the objective of obtaining new genotypes resistant to both diseases simultaneously, two crosses between the apricot selections ‘5–26’ and ‘11–1’ from CEBAS-CSIC (resistant to PPV) and ACLSV-resistant cultivars ‘Mauricio’ and ‘Bergeron’ were achieved, and the descendants were evaluated for resistance to these diseases. Molecular markers were also used to evaluate the genitors and descendants. Results showed that 100% and 75% of seedlings were resistant to PPV and ACLSV, respectively, and 70% were resistant to both diseases simultaneously. In general, no interaction between both viruses was detected, infecting the plant independently. For the first time, apricot plants resistant to PPV and ACLSV simultaneously have been obtained.

Occurrence of cherry viruses in South Tyrol (Italy) by comparing growth periods in two consecutive years

Sweet cherries (Prunus avium L.) are important as fruit crops, and can be affected by numerous viruses. An investigation on the occurrence of the three most common viruses of sweet cherry was carried out in commercially managed orchards in South Tyrol (Italy). The incidence of apple chlorotic leaf spot virus (ACLSV), Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) was investigated using enzyme-linked immunosorbent assays (ELISA) and the reverse transcriptase-polymerase chain reaction technique (RT-PCR) in spring 2018 and 2020, and during the summer and autumn of 2020. All three viruses were detected in the surveyed orchards. Comparative analyses showed that detection was more effective with RT-PCR than with ELISA, especially for detecting PNRSV and PDV. Mixed infections were detected in all the surveyed orchards. The results also showed clear differences between and during host growth periods, likely due to a variable virus concentration in the host trees.

Detection and genetic variation analysis of apple chlorotic leaf spot virus and apple stem grooving virus in wild apple (Malus sieversii) from Xinjiang, China

AbstractHere, we produced nucleotide and amino acid sequences of the movement protein (MP) and the coat protein (CP) genes of apple chlorotic leaf spot virus (ACLSV) and apple stem grooving virus (ASGV) from wild apple of Xinjiang, the main progenitor of the domesticated apple. Genetic variation and phylogenetics were assessed using these and previously reported sequences. Phylogenetic analysis of ACLSV nucleotide and amino acid data sets assigned some ACLSV variants to known groups and others to novel clades. In an MP‐based phylogenetic tree, ASGV sequences downloaded from GenBank and those of wild apple isolates were assigned to separate groups. A recombinant sequence was identified in a ACLSV variant (MS‐GL‐1‐c‐3) of wild apple, and both parents of the recombinant had been isolated from the same plant (GL). Two potential recombination events of ASGV (MS‐GL‐2‐g‐3 and MS‐NF‐1‐g‐3) were identified in this study. These findings may constitute a valuable basis for future studies on variability of virus genetics and pathogenicity.

Evaluation of Apple Chlorotic Leaf Spot Virus Impact on Fruit Yield and Vegetative Growth in Apple Cultivars ‘Antonovka’ and ‘Gita’

Abstract The negative influence of pathogens on plant development is well known. However, the negative impact depends on various factors: cultivar and rootstock resistance, nutrition, the environment, the virulence, and concentration of pathogens in the plant tissue. The evaluation of apple chlorotic leaf spot virus (ACLSV) influence on apple tree growth and production was performed at Institute of Horticulture (LatHort) from 2012 to 2020. Virus-free and ACLSV-infected trees from the traditional cultivar ‘Antonovka’ and new scab resistant cultivar ‘Gita’ were evaluated on dwarfing rootstock B.396. The presence of four common apple viruses (apple mosaic virus (ApMV), ACLSV, apple stem grooving virus (ASGV), apple stem pitting virus (ASPV)) was assayed in the planting year and repeated at the end of the study period. The vigour of planting material, growth, yield, and yield efficiency were compared between virus negative and ACLSV-infected trees. During eight growing seasons (2013–2020), no statistically significant negative impact of ACLSV infection on tree growth or yield was observed for the cultivars. A larger canopy volume was observed for ACLSV-infected trees (p = 0.01), but the cumulative yield per canopy volume was equivalent between virus-negative trees and ACLSV-infected trees (p = 0.07).

Assessment of the virus infections occurrence in new established plum and sweet cherry orchards in Transylvania, Romania

Sixteen plum and eleven sweet cherry newly established commercial orchards in Transylvania, Romania, were the subject of the survey during the vegetative periods of 2020-2021 in order to assess the occurrence of different virus infections. Two blocks of 200 trees from each orchard were monitored by visual observation for virus-like symptoms, especially for Plum pox virus (PPV) in plum orchards. Twenty trees of each plum orchard were then sampled and tested for serological detection of PPV, Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Myrobalan latent ringspot virus (MLRSV). Similarly, ten trees of each sweet cherry orchard were sampled and tested for the presence of PDV, PNRSV, ApMV, ACLSV, PPV, Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Raspberry ringspot virus (RpRSV), Strawberry latent ringspot virus (SLRSV) and Tomato black ring virus (TBRV) by serologic assays. Additionally, a few sweet cherry trees suspected to be infected by Little cherry virus-1 (LChV-1) were tested by molecular assay. Unexpectedly, no plum orchards were found to be free of PPV. The average level of PPV infection was 32%. PNRSV occurred in 8.1% and PDV in 1.2% of sampled plum trees. Four out of ten viruses were detected in sweet cherry sampled: PDV (3.6%), ACLSV (0.9%), RpRSV (0.9%) and TBRV (0.9%). No infection with ACLSV, ApMV and MLRSV in plum and PPV, PNRSV, ApMV, ArMV, CLRV, SLRSV and LChV-1 in sweet cherry was detected. Mixed infections occurred at 4.8% in plum, and at 0.9% in sweet cherry trees sampled. The average occurrence of viruses in plum and sweet cherry orchards surveyed in Transylvania was determined at 41.3%, and 6.3% respectively. Overall results revealed a critical situation especially in regards to PPV infections making the success of fruit production in the most surveyed plum orchards quite problematic. Virus infections level in the younger sweet cherry orchards were significantly lower.

Development and characterization of eco-friendly biopolymer gellan gum based electrolyte for electrochemical application

Abstract Magnesium ion conducting eco-friendly biopolymer electrolyte based on gellan gum has been developed by solution casting technique and characterized by XRD, FTIR, DSC, AC impedance analysis and LSV. Amorphous nature of the polymer electrolyte has been confirmed by XRD analysis. FTIR analysis confirms the complex formation between gellan gum and magnesium nitrate salt. Glass transition temperature of the polymer electrolytes have been found in DSC analysis. Ionic conductivity of polymer electrolyte membrane has been analysized by AC impedance studies, polymer electrolyte 1.0 g gellan gum with 0.7 wt% Mg (NO3)2 has highest ionic conductivity 1.392 × 10−2 S/cm at room temperature. Evan’s polarization method attributes Mg+ cationic transference number as 0.342 for high conducting polymer electrolyte. The high conducting polymer membrane has electrochemical stability 3.58 V. Using this high conducting polymer electrolyte, magnesium ion battery is constructed and the battery performance was studied. The open circuit voltage is found as 1.99 V.

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica).

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.

Apple cultivars and rootstocks assay for the identification of diverse viruses and healthy genotypes for breeding

The prevalence of harmful viruses, viz., apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), and tomato ringspot virus (ToRSV) in apple tree plantations in the Ryazan, Yaroslavl, and Moscow regions, Russian Federation, based on genotype features, planting type (industrial, collection, and repository), and tree age was studied during 2018–2021. The prevalence of harmful and latent viruses ranged from 49.6% to 53.8% in apple cultivars and from 8.3% to 100% in apple clonal rootstocks. The most common virus in the Moscow and Yaroslavl regions was ACLSV (34.7% and 53.8%) and that in the Ryazan region was ApMV (33.3%). Relative to that in young apple trees, the virus influence in older tree tissues was higher by 20% to 43% depending on virus type. Monoviral infection prevailed (59% of all trees were infected with one virus) in the studied apple tree cultivars. Of the cultivars, 25% were infected by a complex of two viruses (ASPV + ACLSV), 10% were infected by three viruses, and 6% were infected by four viruses. The highest prevalence of latent viruses was observed in old Russian cultivars (53.5%) and selections from old foreign cultivars (57.2%). Virus occurrence was slightly lower (51.6%) in new Russian cultivars than in other cultivars. Columnar apple tree cultivars had the lowest virus occurrence (30%) and were found to be more tolerant than other genotypes. The highest virus incidence was recorded in industrial orchards (63.4%), followed by that recorded in collection (20.8%) and repository (18.3%) plantations. By using ELISA, the apple genotypes that were free from harmful viruses were identified as source plants, i.e., 183 plants from 18 apple tree cultivars and 131 plants from clonal rootstocks. Results indicated that virus-free apple germplasm is highly effective for the successful implementation of breeding and genetic improvement.

Seed Transmission of Three Viruses in Two Pear Rootstock Species Pyrus betulifolia and P. calleryana.

Viral seed transmission causes the spread of many plant viral diseases. Pyrus betulifolia and P. calleryana are important rootstock germplasms for pear production in China. This study revealed the widespread infection of apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and apple stem pitting virus (ASPV) in maternal trees of P. betulifolia and P. calleryana by nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assays. Seeds from eight P. betulifolia and two P. calleryana trees had positive rates of 15.9–73.9%, 0–21.2%, and 40.4% for ASGV, ASPV, and ACLSV, respectively. At the cotyledon and 6–8 true leaf stages, seedlings grown from seeds of infected trees gave positive rates of 5.4% and 9.3% for ASGV, 6.7% and 15.6% for ACLSV, and 0% and 2.7% for ASPV, respectively. Incidence in nursery P. betulifolia seedlings of 10.1%, 5.3%, and 3.5% were determined for ASGV, ACLSV, and ASPV, respectively. The nucleotide sequences of coat protein (CP) and movement protein coding genes of both ASGV and ASPV, and CP gene of ACLSV from maternal trees, seeds, and seedlings were analyzed. Sequence identities and phylogenetic comparison with corresponding sequences from GenBank demonstrated that molecular variation occurred within ASGV, ACLSV, and ASPV isolates, with most sequences determined here had close relationships with reported isolates infecting pear or formed independent clades. This is the first report on the seed transmission and the molecular characteristics of these viruses infecting two rootstock species. These findings provided important evidence in management effort for pear viral diseases.

High-Throughput RNA Sequencing of Mosaic Infected and Non-Infected Apple (Malus × domestica Borkh.) Cultivars: From Detection to the Reconstruction of Whole Genome of Viruses and Viroid.

Many viruses have been found associated with apple mosaic disease in different parts of the world. In order to reveal and characterize the viruses and viroids in symptomatic apple plants, next-generation sequencing (RNA seq.) of rRNA-depleted total RNA using Illumina Hiseq2500 was applied to two cultivars, Oregon Spur and Golden Delicious, with symptoms of mosaic and necrosis and one cultivar, Red Fuji, which was asymptomatic. The RNA sequencing detected five viruses, viz., apple necrotic mosaic virus (ApNMV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), and one viroid i.e., apple hammerhead viroid (AHVd). RT-PCR amplification and sequencing also confirmed the presence of all these five viruses and viroids detected in HTS of total RNA. The complete genomes of five viruses and AHVd were reconstructed. The phylogenetic analysis of these viruses and AHVd revealed genetic diversity by forming subclusters with isolates from other countries. Recombination events were observed in all five viruses while single-nucleotide variants were detected only in ApMV and ApNMV. The absence of ApMV and ApNMV in asymptomatic samples from the same cultivars in an RT-PCR assay indicated that these two viruses are associated with mosaic disease of apples in India. This is the first viral genome analysis of symptomatic and asymptomatic apple plants and the first report of genome characterization of viruses associated with apple mosaic disease from India. High-throughput RNA sequencing is a powerful tool to characterize the genome of viruses and viroids in plants previously undetected by conventional methods. This would also help in the indexing and certification of large-scale germplasm.