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

Abstract 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.

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Gospodarski važni virusi jezgričavog voća

Pomologia Croatica ◽

10.33128/pc.22.3-4.5 ◽

2019 ◽

Vol 22 (3-4) ◽

pp. 123-136

Author(s):

Dario Ivić

Keyword(s):

Mosaic Virus ◽

Leaf Spot ◽

Spot Virus ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Chlorotic Leaf ◽

Apple Stem Pitting Virus ◽

Stem Pitting

Virusi jabuke, kruške ili dunje relativno su slabo poznati stručnjacima i voćarima. Najvažnijim virusima koji se javljaju na jezgričavim voćnim vrstama smatraju se virus mozaika jabuke (Apple mosaic virus, ApMV), virus klorotične pjegavosti lista jabuke (Apple chlorotic leaf spot virus, ACLSV), virus brazdavosti debla jabuke (Apple stem grooving virus, ASGV) i virus jamičavosti debla jabuke (Apple stem pitting virus,ASPV). U radu je ukratko opisana njihova važnost, biologija i regulativni status, kao i osnovne mjere zaštite.

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Survey for viruses and viroids in apple and pear trees in New Zealand

New Zealand Plant Protection ◽

10.30843/nzpp.2010.63.6596 ◽

2010 ◽

Vol 63 ◽

pp. 281-281

Author(s):

M.B. Horner

Keyword(s):

New Zealand ◽

Spot Virus ◽

Tomato Ringspot Virus ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Chlorotic Leaf ◽

Pear Trees ◽

Leaf Virus ◽

Stem Pitting ◽

Geographical Locations

Surveys to determine the phytosanitary status of apple (Malus) and pear (Pyrus) trees in New Zealand were conducted from 2005 to 2006 A total of 188 symptomatic and nonsymptomatic trees from various geographical locations were tested for the presence of a number of viruses and viroids by RTPCR All Malus samples were tested for Apple chlorotic leaf spot virus (ACLSV) Apple stem grooving virus (ASGV) Apple stem pitting virus (ASPV) Apple mosaic virus (ApMV) Apple scar skin viroid (ASSVd) Apple dimple fruit viroid (ADFVd) Cherry rasp leaf virus (CRLV) and Tomato ringspot virus (ToRSV) ACLSV was detected in 48 ASGV in 36 ASPV in 61 and ApMV in 45 of samples tested ASSVd ADFVd CRLV ToRSV were not detected in any of the 165 sampled Malus plants which provides evidence that they are not present in New Zealand All Pyrus samples were tested for ACLSV ASGV ASPV ApMV Pear latent virus (PeLV) and Pear blister canker viroid (PBCVd) ACLSV was detected in 17 ASGV in 13 ASPV in 17 and ApMV in 4 of sampled Pyrus trees PeLV and PBCVd were not detected in any of the 23 sampled symptomatic Pyrus trees which provides evidence that PeLV and PBCVd are not present in New Zealand

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First Report of Apple Top Working Disease Caused by Viruses (Apple stem grooving virus, Apple chlorotic leaf spot virus, and Apple stem pitting virus) in Apple in India

Plant Disease ◽

10.1094/pdis-11-12-1082-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 1001-1001 ◽

Cited By ~ 6

Author(s):

A. Brakta ◽

P. D. Thakur ◽

A. Handa

Keyword(s):

Leaf Spot ◽

Spot Virus ◽

Sequence Identity ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Commercial Cultivars ◽

Chlorotic Leaf ◽

Apple Stem Pitting Virus ◽

Stem Pitting ◽

Das Elisa

Top working of apple (Malus domestica Borkh.) trees of old, unproductive, and less preferred cultivars with the newly introduced spur type commercial cultivars has become a common practice with many growers in the northwestern Himalayan region of India. Typical viral symptoms of curling, puckering, and necrosis on leaves were observed with an incidence of 80% on Red Chief, Super Chief, Scarlet Spur, Schillet Spur, Washington Red Delicious, and many other newly introduced cultivars during surveys conducted in May and June 2009. Leaf samples from top worked trees were tested for the presence of Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), and Apple stem pitting virus (ASPV) by employing biological detection (herbaceous and woody indicators), double antibody sandwich (DAS)-ELISA), and reverse transcriptase (RT)-PCR based detection. Mechanical transmission to herbaceous indicators produced chlorotic lesions on Chenopodium quinoa and C. amaranticolor, whereas marginal necrosis was induced on Phaseolous vulgaris within 9 to 21 days after sap inoculations. All three viruses, i.e., ASGV, ASPV, and ACLSV, were detected from these herbaceous indicators in DAS-ELISA (BIOREBA AG, Switzerland). Furthermore, symptoms similar to those observed in orchards were produced when the test budwood was inoculated onto the woody indicator (M. pumila ‘Spy 227’) plant by double grafting, grafting cum budding, and double budding methods within time periods ranging from 4 months in double grafting, 5 months in double budding, to 1 year 4 months in the grafting cum budding method. The presence of all three viruses was confirmed by DAS-ELISA again in Spy 227 woody indicator. PCR detection was carried out by using the coat protein gene specific primers (ASGV5641 [forward], ASGV6396 [reverse]; ACLSV6784 [forward], ACLSV7365 [reverse] [2]; ASP-C [sense], ASP-A [anti-sense] [1]) of all the viruses detected through ELISA. The amplified products were cloned, sequenced, and deposited in NCBI (GenBank Accessions KC110892 for ASGV, KC154859 for ASPV, and KC154862 for ACLSV). BLASTn analysis showed the ASGV isolate had 97 to 98% sequence identity with Indian (FM204881) and Brazilian (AF438409) ASGV isolates. The ASPV and ACLSV isolates had 98% and 99% sequence identity with Chinese (JF895517) and Japanese (AB326230) isolates, respectively. To the best of our knowledge, this is the first report of apple top working disease associated with ASGV, ASPV, and ACLSV infection in commercial cultivars of apple from India and seems to be a serious threat for growing virus-free healthy stocks in orchards. Top working disease in apple associated with ASGV, ASPV, and ACLSV viruses has been reported from Japan (3,4). References: (1) J. K. Kundu et al. Plant Prot. Sci. 39:88, 2003. (2) O. Nickel et al. Fitopatol. Brasil. 26:655, 2001. (3) H. Yanase. Bull. Fruit Tree Res. Stn., Japan Ser. C 1:47, 1974. (4) H. Yanase et al. Acta Hortic. 44:221, 1975.

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First Report of Apple stem grooving virus, Apple stem pitting virus, and Apple chlorotic leaf spot virus in Apple Trees From Chencha, Ethiopia

Plant Disease ◽

10.1094/pdis-05-16-0678-pdn ◽

2016 ◽

Vol 100 (12) ◽

pp. 2540 ◽

Cited By ~ 2

Author(s):

B. Lemma ◽

F. Xing ◽

S. F. Li ◽

Z. X. Zhang

Keyword(s):

Leaf Spot ◽

Apple Trees ◽

Spot Virus ◽

First Report ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Chlorotic Leaf ◽

Apple Stem Pitting Virus ◽

Stem Pitting

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PECULIARITIES OF BIOECOLOGY OF HARMFUL VIRUSES IN APPLE PLANTS

POMICULTURE & SMALL FRUITS CULTURE IN RUSSIA ◽

10.31676/2073-4948-2021-64-93-100 ◽

2021 ◽

Vol 64 ◽

pp. 93-100

Author(s):

M. Т. Upadyshev ◽

A. D. Petrova ◽

E. A. Tut'

Keyword(s):

Apple Tree ◽

Sandwich Elisa ◽

Apple Rootstocks ◽

Spot Virus ◽

Serological Tests ◽

Apple Varieties ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Chlorotic Leaf ◽

Stem Pitting

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

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Simultaneous detection of four viruses affecting apple and pear by molecular hybridization using a polyprobe

Ciência Rural ◽

10.1590/0103-8478cr20131629 ◽

2014 ◽

Vol 44 (10) ◽

pp. 1711-1714 ◽

Cited By ~ 5

Author(s):

Thor Vinícius Martins Fajardo ◽

Osmar Nickel

Keyword(s):

Leaf Spot ◽

Simultaneous Detection ◽

Hybridization Method ◽

Spot Virus ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Chlorotic Leaf ◽

Viral Cdna ◽

Apple Stem Pitting Virus ◽

Stem Pitting

The viruses Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV) and Apple mosaic virus (ApMV) are common in apples and pears and main targets of detection in propagation materials. This study aimed at demonstrating the usefulness of the hybridization method with a non-radioactive probe for simultaneous detection of these four viruses. The sensitivity of this method was sufficiently high enabling the detection of ASGV, ACLSV, ASPV and ApMV in total RNA extracted from infected samples. The probe specificity was confirmed by reaction with homologous viral cDNA, individually cloned for each virus.

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Effect of Thermotherapy on Elimination of Apple Stem Grooving Virus and Apple Chlorotic Leaf Spot Virus for In vitro-cultured Pear Shoot Tips

HortScience ◽

10.21273/hortsci.41.3.729 ◽

2006 ◽

Vol 41 (3) ◽

pp. 729-732 ◽

Cited By ~ 27

Author(s):

Liping Wang ◽

Guoping Wang ◽

Ni Hong ◽

Rongrong Tang ◽

Xiaoyun Deng ◽

...

Keyword(s):

Leaf Spot ◽

Blot Hybridization ◽

Shoot Tips ◽

Dot Blot ◽

Spot Virus ◽

Dot Blot Hybridization ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Chlorotic Leaf

Apple stem grooving virus (ASGV) and apple chlorotic leaf spot virus (ACLSV) are two major viruses of pear. In this study, in vitro thermotherapy was carried out at 37°C for 25, 30 and 35 days followed by subculturing of meristem tips of different sizes to eliminate ASGV and ACLSV from pear plants. Virus titers in heat-treated shoot tips were evaluated by ELISA testing of regenerated plants. Results showed that thermotherapy for 35 days significantly decreased the titer of ASGV and ACLSV in cultures regenerated from tips of main and axillary shoots, especially in those from explants 1 mm in length from the tip of meristems. Dot-blot hybridization of biotinylated cDNA probes derived from ACLSV and ASGV was used to detect these viruses in crude tissue extracts of in vitro-grown pear plants. Intense signals were consistently detected in untreated plant samples equivalent to less than 0.5 mg tissue. Comparison of signals from dot-blot hybridization and ELISA absorbance values (A405) confirmed that dot-blot hybridization had a higher sensitivity than PAS-ELISA. Dot-blot hybridization could detect viruses with a titer below the threshold level of ELISA. These results indicate that dot-blot hybridization is a useful tool for large-scale surveys of viruses, which facilitates the production of virus-free propagation materials in certification and sanitation programs. Results of PAS-ELISA and dot-blot hybridization showed that high virus elimination efficiency was achieved by a combination of thermotherapy for 35 days and in vitro culture of 1 mm meristem tips.

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