Vitrification Cryo-Foil for Apple Cryopreservation and the Seesaw Effect Between Shoot Recovery and Virus Eradication

Improvements of existing cryopreservation protocols are necessary to facilitate long-term preservation of plant germplasm and the cryotherapy-effect of pathogen eradication. This study reported a vitrification (V) cryo-foil/plate methods for cryopreservation of shoot tips and cryotherapy effect in ‘Pink Lady’ apple. In V cryo-foil/plate protocols, shoot tips were first attached onto aluminum foils/plates using calcium alginate before other procedures. Shoot tips cryopreserved by V cryo-foil required 6.1 weeks to fully recover and 53% of shoot regrowth was obtained, comparable to the Dv cryopreservation. Similar regrowth levels were produced between applying V cryo-foil and Dv cryopreservation to another 4 Malus genotypes. Histological observations in shoot tips cryopreserved by Dv and V cryo-foil found only those with surviving apical dome and leaf primordia (LPs) could recover after cryopreservation. In apical meristem of shoot tips cryopreserved by Dv and V cryo-foil, higher surviving probability was detected from the V cryo-foil protocol, and the young LPs showed the highest level of surviving. Virus detection in cryo-derived plants showed apple stem grooving virus and apple chlorotic leaf spot virus were all preserved after cryopreservation, and higher eradication efficiency of apple stem pitting virus (70%) was produced by Dv than the 55% of V cryo-foil. These results supported applying V cryo-foil as an improvement to the widely applied Dv method in shoot tip cryopreservation, and also revealed a seesaw mode between shoot recovery and cryotherapy effect. Once the seesaw moves to increase the recovery after cryopreservation, the cryotherapy-effect on the other side would be decreased.

First Detection and Molecular Characterization of Apple Stem Grooving Virus, Apple Chlorotic Leaf Spot Virus, and Apple Hammerhead Viroid in Loquat in Spain

Loquat (Eriobotrya japonica) is an important crop in Spain. To date, only one viral species, apple stem pitting virus (ASPV), has been detected in Spanish loquat orchards. In this study, the presence of additional viruses infecting this crop in Spain was investigated. RT-PCR and high-throughput sequencing (HTS) of symptomatic loquat plants led to first-time detection and characterization of apple stem grooving virus (ASGV), also known as citrus tatter leaf virus (CTLV), and apple chlorotic leaf spot virus (ACLSV) from Spain with description of nearly complete genomic sequences. The frequency of ACLSV infection was the highest, with over 30% of the samples testing positive and were also detected as coinfections with ASGV and ASPV, although most of the samples infected were symptomless. Studies on all the full-length sequences available in the databases were performed in order to establish the phylogenetic relationships of the Spanish isolates of these two viral species. Moreover, apple hammerhead viroid (AHVd) was also detected to infect loquat, the first host different from apple reported for this viroid to date.

Characterisation and Distribution of Karaka Ōkahu Purepure Virus—A Novel Emaravirus Likely to Be Endemic to New Zealand

We report the first emaravirus on an endemic plant of Aotearoa New Zealand that is, to the best of our knowledge, the country’s first endemic virus characterised associated with an indigenous plant. The new-to-science virus was identified in the endemic karaka tree (Corynocarpus laevigatus), and is associated with chlorotic leaf spots, and possible feeding sites of the monophagous endemic karaka gall mite. Of the five negative-sense RNA genomic segments that were fully sequenced, four (RNA 1–4) had similarity to other emaraviruses while RNA 5 had no similarity with other viral proteins. A detection assay developed to amplify any of the five RNAs in a single assay was used to determine the distribution of the virus. The virus is widespread in the Auckland area, particularly in mature trees at Ōkahu Bay, with only occasional reports elsewhere in the North Island. Phylogenetic analysis revealed that its closest relatives are pear chlorotic leaf spot-associated virus and chrysanthemum mosaic-associated virus, which form a unique clade within the genus Emaravirus. Based on the genome structure, we propose this virus to be part of the family Emaravirus, but with less than 50% amino acid similarity to the closest relatives in the most conserved RNA 1, it clearly is a novel species. In consultation with mana whenua (indigenous Māori authority over a territory and its associated treasures), we propose the name Karaka Ōkahu purepure virus in te reo Māori (the Māori language) to reflect the tree from which it was isolated (karaka), a place where the virus is prevalent (Ōkahu), and the spotted symptom (purepure, pronounced pooray pooray) that this endemic virus appears to cause.

Black Leg and Chlorotic Leaf Spot Occurrence on Brassicaceae Crop and Weed Hosts

Black leg (caused by Plenodomus lingam and P. biglobosus) and chlorotic leaf spot (caused by Pyrenopeziza brassicae) are economically important fungal diseases of Brassicaceae crops. Surveys of seed fields and weed hosts were conducted to understand the distribution and prevalence of these diseases in Oregon after black leg and chlorotic leaf spot outbreaks occurred in Brassicaceae crops in 2014. Post-harvest black leg ratings for these diseases were conducted in 2015 and 2016 in seed fields of canola, forage rape, and turnip. Black leg incidence was greater in turnip (51%) compared to canola (29%) and forage rape (25%). The overall average disease incidence was greater on seed crops harvested in 2015 (46%) compared to crops harvested in 2016 (28%). A disease survey of wild Brassicaceae plants was conducted along Interstate 5 in Oregon. Brassicaceae weed population sites were identified and 40 sites were sampled for these diseases. Black leg and chlorotic leaf spot were present in 60% and 45%, respectively, of the sampled sites. Both species of Plenodomus were detected in weed populations with P. lingam being the predominant species recovered (95%). The northernmost sample site with black leg was found less than 32 km from the Oregon-Washington border, and southernmost site occurred within 32 km of the Oregon-California border. Chlorotic leaf spot was detected less than 32 km from Oregon-Washington border, whereas the southernmost detection was approximately 164 km from the Oregon-California border. Based on this study, infected crop residues and weed hosts may facilitate persistence and spread of these pathogens.

PECULIARITIES OF BIOECOLOGY OF HARMFUL VIRUSES IN APPLE PLANTS

The prevalence of harmful viruses of Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), depending on the location, age, and varietal composition of plantings, was studied. Sandwich ELISA was used in serological tests. Diagnostic kits from «Loewe» (Germany) were used for the analyzes. Leaves were taken as samples. The results of the analysis were recorded using a «Stat Fax 2100» photometer. On apple varieties and clonal rootstocks, the frequency of viruses was 36-54 % and 24 %, respectively. In the Moscow region, the ASPV virus (23 %) prevailed, in the Ryazan region — ApMV (33 %), in the Yaroslavl region — ACLSV (54 %). At the end of the 20th century, the ACLSV virus was more often detected on the apple tree, and in the last decade, we have established an increase in the infection of this crop with the more harmful ASPV and ASGV viruses. The highest prevalence of latent viruses was noted on old domestic apple varieties and foreign varieties (50 % each). A tendency to an increase in the indices of infection with most of the studied viruses was revealed with an increase in the age of trees. In apple trees of old age (over 20 years), the indices of infection with the ASPV virus increased by 25 %, ASGV — by 20 %, ACLSV — by 43 % compared to middle-aged trees (8-10 years). Apple plants of 15 varieties and apple rootstocks of 2 forms 54-118 and 57-490 were found free from the main harmful viruses

The begomovirus species Melon chlorotic leaf curl virus is composed of two highly divergent strains that differ in their genetic and biological properties

Since the early 1990s, squash production in Costa Rica has been affected by a whitefly-transmitted disease characterized by stunting and yellow mottling of leaves. The squash yellow mottle disease (SYMoD) was shown to be associated with a bipartite begomovirus, originally named squash yellow mild mottle virus (SYMMoV). It was subsequently established that SYMMoV is a strain of melon chlorotic leaf curl virus (MCLCuV), a bipartite begomovirus that causes a chlorotic leaf curl disease of melons in Guatemala. In the present study, the complete sequences of the DNA-A and DNA-B components of a new isolate of the strain MCLCuV-Costa Rica (MCLCuV-CR) were determined. Comparisons of full-length DNA-A sequences revealed 97% identity with a previously characterized isolate of MCLCuV-CR, and identities of 90 to 91% with those of isolates of the strain MCLCuV-Guatemala (MCLCuV-GT), which is below or at the current begomovirus species demarcation threshold of 91%. A more extensive analysis of the MCLCuV-CR and -GT sequences revealed substantial divergence in both components and different histories of recombination for the DNA-A components. The cloned full-length DNA-A and DNA-B components of this new MCLCuV-CR isolate were infectious and induced SYMoD in a range of squashes and in pumpkin, thereby fulfilling Koch’s postulates for this disease. However, in contrast to MCLCuV-GT, MCLCuV-CR induced mild symptoms in watermelon and no symptoms in melon and cucumber. Taken together, our results indicate that MCLCuV-CR and -GT have substantially diverged, genetically and biologically, and have evolved to cause distinct diseases of different cucurbit crops. Taxonomically, these viruses are at the strain/species boundary, but retain the designation as strains of Melon chlorotic leaf curl virus under current ICTV guidelines.