Characterization of the Mycovirome of the Phytopathogenic Fungus,Neofusicoccum parvum

Neofusicoccum parvum is a fungal plant-pathogen belonging to the family Botryosphaeriaceae, and is considered one of the most aggressive causal agents of the grapevine trunk disease (GTD) Botryosphaeria dieback. In this study, the mycovirome of a single strain of N. parvum (COLB) was characterized by high throughput sequencing analysis of total RNA and subsequent bioinformatic analyses. Contig annotations, genome completions, and phylogenetic analyses allowed us to describe six novel mycoviruses belonging to four different viral families. The virome is composed of two victoriviruses in the family Totiviridae, one alphaendornavirus in the family Endornaviridae, two mitoviruses in the family Mitoviridae, and one narnavirus belonging to the family Narnaviridae. The presence of the co-infecting viruses was confirmed by sequencing the RT-PCR products generated from total nucleic acids extracted from COLB. This study shows that the mycovirome of a single N. parvum strain is highly diverse and distinct from that previously described in N. parvum strains isolated from grapevines.     

Genetic diversity and phylogenetic analysis of Australian Grapevine Viroid (AGVd) isolated from different grapevines in China

Australian grapevine viroid (AGVd) is found in only three countries in the world. Here, the genetic diversity and phylogenetic relationships of AGVd isolates from three different grape varieties (Thomson Seedless, Jingchuan and Zaoyu) in China were studied. A hundred of independent cDNA clones from each of the three isolates, in total of 300, were sequenced. We identified 29 sequence variants including two predominant ones in Thomson Seedless, and 48 each including a unique predominant one in Jingchuan and Zaoyu. In silico structure analysis revealed that base changes were clustered in the left terminal domain of the predicted secondary structure in all three isolates. Further, these changes were shown to affect their secondary structures to varying degrees. Genetic diversity and phylogenetic analysis of four predominant sequence variants from this study, plus four others from Australia and Tunisia, revealed obvious regional disparity and variety-specificity in AGVd.     

新疆吐鲁番、喀什葡萄树伤流液多糖分离纯化及其抗氧化活性比较研究

目的确定新疆葡萄树伤流液多糖含量,探究吐鲁番无核白和喀什木纳格萄萄树伤流液多糖及其纯化组分的抗氧化活性。方法采用蒽酮-硫酸法检测多糖含量,利用醇沉法和改进的Sevage法制备粗多糖,经过DEAE-52纤维素柱对粗多糖进行分离纯化,以羟自由基、超氧自由基、DPPH·自由基、亚硝基自由基、ABTS·自由基为指标检测多糖组分及原液的抗氧化能力。结果吐鲁番无核白和喀什木纳格葡萄树伤流液中多糖含量分别为114和193μg·mL-1,经过DEAE-52纤维素柱分离分别得到8个多糖组分,其中两者4个含量较高的多糖组分和原液都具有不同程度的抗氧化活性,且对羟自由基的清除能力强于对其他4种自由基的清除能力,两地葡萄树伤流液的抗氧化活性存在差异。结论吐鲁番无核白和喀什木纳格葡萄树伤流液的多糖组分均具有一定的抗氧化活性,并且其抗氧化活性存在差异。     

Toxic Responses and Catalase Activity ofLemna minorL. Exposed to Folpet, Copper, and Their Combination

Toxicity of copper and folpet—two fungicides widely used on grape—was evaluated onLemna minorL., a sensitive aquatic weed regularly used for (eco)toxicological studies. Toxicity assessments were based on inhibition of growth and chlorophyll content ofL. minorcultures after 7 days. IC₁₀, IC₅₀, and IC₉₀were determined for both compounds alone and were respectively, 0.03, 0.16, and 0.95 mg liter⁻¹for copper and 1.20, 7.50, and >40 mg liter⁻¹for folpet. When both compounds were combined, the response ofL. minordepended on the initial folpet concentration. Indeed, a slight synergy was observed for 5 mg liter⁻¹folpet, while at folpet concentrations of 20 to 35 mg liter⁻¹, the two fungicides were antagonists. The antagonism was positively correlated with folpet concentration. Antagonism between Cu and folpet could not be explained by a reduced bioavailability of Cu since concentration of free copper in the mixture did not depend on the presence of folpet. One physiological defense response elicited by copper in plants is an increase in catalase activity. Copper and folpet stimulated catalase activity and changes in the activity of the enzyme could not account for the synergy but possibly for the antagonism. Nevertheless, catalase activity increased significantly after a 24-h exposure to 25 μg liter⁻¹of copper. The use of this property as a rapid and sensitive biomarker to monitor the toxicity of xenobiotics alone or in combination and of environmental water is discussed.     

Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics

Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.     

新疆葡萄树伤流液蛋白含量测定及抗氧化活性研究

目的了解新疆葡萄树伤流液蛋白含量及其抗氧化能力。方法采用考马斯亮蓝法测定新疆葡萄树伤流液的蛋白含量;以抗坏血酸为阳性对照,应用ABTS法、羟A由基试剂盒法、DPPH法和亚硝酸盐法,研究其对自由基的清除能力。结果不同产地、品种的葡萄树伤流液蛋白含量不同,其中和田木纳格最高,为22．4±0．07μg／mL;不同产地、品种的葡萄树伤流液清除自由基的能力不同,但对·OH的清除能力明显高于对DPPH·和NO2^-·的清除能力。结论新疆葡萄树伤流液因产地和品种的不同,其蛋白含量及清除自由基能力也不同。     

Phylogenetic Analysis of Hop and Grapevine Isolates of Hop Stunt Viroid Supports a Grapevine Origin for Hop Stunt Disease

We have examined sequence variability among nine isolates of hop stunt viroid (HSVd) collected from hop gardens in Tohoku district in Japan, the only area in the world where hop stunt disease is endemic. Six different consensus and one-consensus sequences as well as 12 sequence variants were detected in the nine HSVd-hop isolates, which suggested the sequence of HSVd-hop was remarkably variable. A neighbor-joining analysis was carried out on the new HSVd-hop sequences together with 44 previously described variants of HSVd isolated from hop and other species. All the HSVd-hop sequences recovered from hops cultivated in the Tohoku district of Japan as well as the type isolate and two Korean isolates form a cluster with the HSVd-g subtype 1 commonly recovered from grapevine. This close relationship between HSVd-hop and -grapevine isolates strongly supports the grapevine origin for hop stunt disease.