Combining Thermotherapy with Shoot Tip Culture or Cryotherapy for Improved Virus Eradication from In Vitro Actinidia macrosperma.

In recent years, kiwifruit viral diseases have become increasingly prevalent in kiwifruit-producing regions of China, significantly impacting both the yield and quality of kiwifruit. This has emerged as a significant constraint on the healthy and sustainable development of the kiwifruit industry. The use of virus-free propagation materials has been proven the most effective strategy for controlling plant viral diseases. In the present study, shoot tip culture, shoot tip cryotherapy, and their combinations with thermotherapy were established to eradicate Actinidia virus A (AcVA), Actinidia virus B (AcVB), and Actinidia chlorotic ringspot-associated virus (AcCRaV) from Actinidia macrosperma. Additionally, the impact of shoot tip size on virus eradication was evaluated. Among the three confirmed viruses, regardless of the procedure, AcVB was the easiest to eradicate, followed by AcVA and AcCRaV. Combining thermotherapy with shoot tip culture or cryotherapy resulted in a higher virus-free frequency (up to 27.3 and 50%, respectively) than shoot tip culture or cryotherapy alone (0 to 20%). Notably, the combination of thermotherapy and 0.5- to 1-mm shoot tip cryotherapy was shown to be the most effective protocol for virus eradication from A. macrosperma, which produced 50% of regenerated shoots free from all the tested viruses. To the best of our knowledge, this is the first report on virus elimination from kiwifruit infected with multiple viruses based on conventional shoot tip culture and shoot tip cryotherapy.

Efficient Elimination of Actinidia Chlorotic Ringspot-Associated Virus from Infected Kiwifruit Shoots Cultured In Vitro.

In this study, methods of Actinidia chlorotic ringspot-associated virus (AcCRaV) elimination by shoot tip culture, thermotherapy followed by shoot tip culture, and chemotherapy followed by shoot tip culture were explored. The results showed that the AcCRaV elimination rate was 23.3% when the secondary shoot tip culture method was used and when the shoot tip length was less than 0.5 mm. The AcCRaV elimination rate was 100% when thermotherapy (36°C [day] and 32°C [night]) was applied for 20 days followed by shoot tip culture (shoot tip length less than 1.0 mm). When shoot segments were treated with ribavirin at 15 µg/ml for 2 months followed by shoot tip culture, the elimination rate of AcCRaV was 100% (shoot tip length less than 1.0 mm). When shoot segments were treated with ribavirin at 25 µg/ml for 2 months followed by shoot tip culture, the elimination rate of AcCRaV was 100% (shoot tip length less than 1.5 mm). This is the first report on kiwifruit virus elimination methods.

Advances in and Prospects for Actinidia Viruses.

Kiwifruit (Actinidia spp.) is an economically important fruit crop worldwide. Before 2010, kiwifruit viruses had not received much attention; since then, more than 20 viruses infecting kiwifruit have been discovered. Some of these viruses cause severe yellowing, mosaic, necrosis, ringspots, and other symptoms on leaves, seriously impacting yield and quality. Many of these viruses are widely distributed. This review summarizes recent research advances in the identification, genomic variation, distribution, transmission, detection, incidence, prevention, and control of kiwifruit viruses and proposes directions for future research. Using virus-tested propagation material is the most economical and effective method for controlling kiwifruit viruses.

Viromics Reveals the Viral Diversity in Cultivated and Wild Kiwifruit.

China, the center of origin of kiwifruit, has the largest kiwifruit cultivation and production area worldwide, and Shaanxi Province is the major kiwifruit-growing region in China. However, our knowledge of kiwifruit viruses is largely skewed toward their pathology in cultivated orchards, and little is known about viral diversity in wild kiwifruit. To determine the viral diversity in cultivated and wild kiwifruit, 32 cultivated kiwifruit samples from Shaanxi Province and 30 wild kiwifruit samples from the Qinling Mountains were collected and subjected to high-throughput sequencing in this study. Eleven known viruses were found among the 32 cultivated kiwifruit samples, and 8 known viruses and 2 new viruses were found among the 30 wild kiwifruit samples. One of the two new viruses, Actinidia yellowing virus 3 (AcYV3), a member of the genus Idaeovirus, may be associated with severe yellowing of kiwifruit leaves. In addition, more than 50 nearly full-length genome sequences of known viruses were obtained. The detection rates, recombination, and molecular variation of these viruses were further analyzed. The results obtained in this study provide valuable information for understanding the virome of cultivated and wild kiwifruit.

Occurrence and Distribution of Actinidia Viruses in Shaanxi Province of China.

Kiwifruit (Actinidia spp.) is an economically important fruit crop globally. China is the largest kiwifruit-growing country in the world, and Shaanxi Province is the major kiwifruit-growing region in China. A systematic survey detected various symptoms in kiwifruit plants grown in a commercial kiwifruit field in Shaanxi Province. Samples were collected from kiwifruit plants showing symptoms and used for virus detection by high-throughput sequencing. In addition to 10 known kiwifruit viruses, three new viruses were detected and tentatively named Actinidia yellowing ringspot virus (AYRSpV), Actinidia yellowing virus 1 (AcYV1), and Actinidia yellowing virus 2 (AcYV2). The genome sequences of the three new viruses and four known viruses were determined. Based on the demarcation criteria of the International Committee on Taxonomy of Viruses, AYRSpV might be a new member of the genus Ilarvirus in the family Bromoviridae, AcYV1 might be a new virus of the genus Waikavirus in the family Secoviridae, and AcYV2 might be a novel virus in the family Tombusviridae. Spherical viral particles were found in the samples infected with AYRSpV, AcYV1, and AcYV2 by transmission electron microscopy. Further analysis showed that all 13 viruses can infect both Actinidia deliciosa and A. chinensis but the incidences of these infections vary among different kiwifruit cultivars in different regions. These results provide valuable information for understanding the virome of kiwifruit in China.

Next-Generation Sequencing Combined With Conventional Sanger Sequencing Reveals High Molecular Diversity in Actinidia Virus 1 Populations From Kiwifruit Grown in China.

Kiwifruit (Actinidia spp.) is native to China. Viral disease-like symptoms are common on kiwifruit plants. In this study, six libraries prepared from total RNA of leaf samples from 69 kiwifruit plants were subjected to next-generation sequencing (NGS). Actinidia virus 1 (AcV-1), a tentative species in the family Closteroviridae, was discovered in the six libraries. Two full-length and two near-full genome sequences of AcV-1 variants were determined by Sanger sequencing. The genome structure of these Chinese AcV-1 variants was identical to that of isolate K75 and consisted of 12 open reading frames (ORFs). Analyses of these sequences together with the NGS-derived contig sequences revealed high molecular diversity in AcV-1 populations, with the highest sequence variation occurring at ORF1a, ORF2, and ORF3, and the available variants clustered into three phylogenetic clades. For the first time, our study revealed different domain compositions in the viral ORF1a and molecular recombination events among AcV-1 variants. Specific reverse transcriptase-polymerase chain reaction assays disclosed the presence of AcV-1 in plants of four kiwifruit species and unknown Actinidia spp. in seven provinces and one city.