Development and detection application of monoclonal antibodies against Zucchini yellow mosaic virus

Aphid-borne Zucchini yellow mosaic virus （ZYMV） is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection technique. Using purified ZYMV virion and the conventional hybridoma technology, three hybridoma cell lines （16A11, 5A7 and 3B8） secreting monoclonal antibodies （MAbs） against ZYMV Zhejiang isolate were obtained. The working titers of the ascitic fluids secreted by the three hybridoma cell lines were up to 10^-7 by indirect enzyme-linked immunosorbent assay （ELISA）. All MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that the MAb 3B8 could specifically react with the coat protein of ZYMV while MAbs 5A7 and 16A11 reacted strongly with a protein of approximately 51 kDa from the ZYMV-infected leaf tissues. According to this molecular weight, we consider this reactive protein As likely to be the HC-Pro protein. Using these three MAbs, we have now developed five detection assays, i.e., antigen-coated-plate ELISA （ACP-ELISA）, dot-ELISA, tissue blot-ELISA, double-antibody sandwich ELISA （DAS-ELISA）, and immunocapture-RT-PCR （IC-RT-PCR）, for the sensitive, specific, and easy detection of ZYMV. The sensitivity test revealed that ZYMV could be readily detected respectively by ACP-ELISA, dot-ELISA, DAS-ELISA and IC-RT-PCR in 1：163840, 1：2560, 1：327680 and 1：1 310720 （w/v, g mL-1） diluted crude extracts from the ZYMV-infected plants. We demonstrated in this study that the dot-ELISA could also be used to detect ZYMV in individual viruliferous aphids. A total of 275 cucurbitaceous plant samples collected from the Zhejiang, Jiangsu, Shandong and Hainan provinces, China, were screened for the presence of ZYMV with the described assays. Our results showed that 163 of the 275 samples （59%） were infected with ZYMV. This finding indicates that ZYMV As now widely present in cucurbitaceous crops in China. RT-PCR followed by DNA sequencing and sequence analyses confirmed the accuracy of the five assays. We consider that these detection assays can significantly benefit the control of ZYMV in China.

Efficacy of the Pepper Extracts to Control Zucchini Yellow Mosaic Virus

The Zucchini yellow mosaic virus （ZYMV） （Potyvirus） on “zucchini” presents great economic importance for Malagasy farmers. Numerous aphid species （Hemiptera： Aphididae） spread viral particles, which are easily transmitted mechanically, too. Farmers ignored this disease, therefore, its control became extremely difficult with insecticides. This study aimed to evaluate efficacy of chili pepper extracts. Treatments vary as a function of its initiations and were repeated at weekly intervals until harvest. It was conducted in market gardens around the town of Antananarivo. The study compared four treatments and repeated three times： firstly, plots received a protection as soon as installation of culture （preventive protection）; secondly, plots received treatment with low infestation （about a quarter plants infested） （late protection）; thirdly, plots received treatment with high infestation （about half of plants infested） （latest protection）; lastly, plots without treatment （the control）. As a result, pepper extracts can provide a significant level to control against aphids attack, and permit to delay virus installation on plots, which received treatment as soon as implementation of culture. However, it cannot eradicate and cannot limit extend of virus disease on plots already infested. Economic analysis shows that preventive treatment of pepper extracts to fight virus attack provides an even greater return on its investment than all other terms. Thus, use of pepper extracts can reduce chemical treatment and pollution.

Responses of Some American, European and Japanese Wheat Cultivars to Soil-Borne Wheat Viruses in China

Wheat seeds of 109 cultivars from USA, Europe and Japan were sown in experiments at seven sites in different provinces of China for one or two seasons. Five of the sites were infested with the bymovirus wheat yellow mosaic virus (WYMV) and two jointly with WYMV and the furovirus Chinese wheat mosaic virus (CWMV). Disease symptoms were assessed visually and leaf samples were tested for virus (es) by ELISA. At least 29 cultivars were resistant to WYMV at the sites where only this virus was present but all the cultivars were severely infected at Rongcheng (Shandong Province) where CWMV was mixed with WYMV. There was evidence that the presence of CWMV assisted infection by WYMV and also resulted in more severe symptoms. At the mixed site in Yantai, Shandong Province, symptoms were mild and many cultivars had symptomless infection . Of the two strains of WYMV identified in Japan, the Chinese sites seem to be most similar to the type isolated, WYMV-T. Eleven cultivars seemed to be susceptible to WYMV only at Loutian (Hubei Province),suggesting that the virus at this site would be worth studying further.

Establishment of Multiplex RT-PCR Detection System for Three Viruses in Freesia

The specific primers were designed according to conserved sequences of coat protein （CP） gene of Freesia mosaic virus （FreMV）, Cucumber mosaic vi- rus （CMV） and Bean yellow mosaic virus （BYMV） published in GenBank, and a multiplex PCR protocol for simultaneous detection of these three viruses in freesia was developed. Three specific fragments were simultaneously amplified in a single PCR reaction. Their lengths were determined to be 340,628 and 212 bp, respec-tively. The sequence analysis indicated that three viruses shared at least 97% of homology with reference sequence. Sensitivity test showed that these three viruses could be detected out in the infected plant tissue greater than 10^-2 mg.

应用F(ab')_2^-酶联吸附分析法检测大麦黄花叶病毒

F(ab′)_2酶联免疫吸附分析法(F(ab′)_2-ELISA)成功地用于大麦黄花叶病毒(BaYMV)的常规检测和诊断.其步骤是先用稀释1000—4000倍的抗血清F(ab′)_2包被反应板,加待测样品和稀释1000倍的同种抗血清或IgG,然后再加A蛋白碱性磷酸酯酶和底物,测定OD值。比较试验表明,ELISA稀释缓冲液加入1％小牛血清或1％全脂奶粉,BaYMV的测检灵敏度可提高达2.5—5.0ng/ml,病叶汁液检测终浓度为稀释1600—3200倍。我国BaYMV分离物与英国分离物的血清学性质完全一致。BaYMV在大麦病株中以叶部含量较高,茎中含量次之,根部测不出病毒。检测和诊断田间样品,即使有的样品已不新鲜,也均能得到满意的结果。此方法也成功地用于大麦温和花叶病毒(BaMMV)、小麦黄花叶病毒(WYMV)、燕麦花叶病毒(OMV)和燕麦金色条纹病毒(OGSV)等禾谷多粘菌传麦毒的检测,这S种病毒的血清学关系研究表明,除BaYMV和WYMV之间具有血清学关系以外,其余彼此均不反应。

An Integrated Approach to Comprehend MYMIV-Susceptibility of Blackgram Cv. T9 Possessing Allele of CYR1, the Cognate R-Gene

Blackgram, an important legume crop, faces the constraint of Mungbean yellow mosaic India virus (MYMIV)-stress resulting in severe crop penalty. MYMIV-resistant plants exhibit incompatible response via a cognate CYR1 gene-mediated interaction with virus effector molecule. In this study, we searched for the susceptible allele of the “R” gene in Cv. T9. Southern hybridization study confirmed presence of an allele in Cv. T9. However, transcripts of the CYR1 could not be detected either by RT-PCR or by Northern hybridization in Cv. T9 and also in other susceptible blackgram line. The allele was isolated, sequenced and referred as cyr1. In silico study revealed that cyr1 also encodes a CC-NBS-LRR protein like CYR1. However the CC domain of cyr1 is truncated by 128 amino acid residues indicating functional impairment with respect to the signal transduction after pathogen invasion. Comparative 3D structural modeling, hydrogen bonding and Van der Waals interaction studies revealed differences between CYR1 and cyr1. Lys519 and Thr490 present in the largest pockets of the CYR1 are the key interacting hotspots between CYR1 and MYMIV coat protein (CP). The weak Van der Waals interactions and intermolecular hydrogen bonding between cyr1 and CP confers less stability to the molecular recognition complex, unlike CYR1. Thus, the present investigation revealed Cv. T9 shows compatible interaction with MYMIV due to the truncation in the cyr1 sequence and consequent structural difference in the N-terminal of CC-domain.

A virus-derived siRNA activates plant immunity by interfering with ROS scavenging

Virus-derived small interference RNAs(vsiRNAs)not only suppress virus infection in plants via induction of RNA silencing but also enhance virus infection by regulating host defensive gene expression.However,the underlying mechanisms that control vsiRNA-mediated host immunity or susceptibility remain largely unknown.In this study,we generated several transgenic wheat lines using four artificial microRNA expression vectors carrying vsiRNAs from Wheat yellow mosaic virus(WYMV)RNA1.Laboratory and field tests showed that two transgenic wheat lines expressing amiRNAI were highly resistant to WYMV infection.Further analyses showed that vsiRNAI could modulate the expression of a wheat thioredoxin-like gene(TaAAEDI),which encodes a negative regulator of reactive oxygen species(ROS)production in the chloroplast.The function of TaAAEDI in ROS scavenging could be suppressed by vsiRNAI in a dose-dependent manner.Furthermore,transgenic expression of amiRNAI in wheat resulted in broad-spectrum disease resistance to Chinese wheat mosaic virus,Barley stripe mosaic virus,and Puccinia striiformis f.sp.tritici infection,suggesting that vsiRNAI is involved in wheat immunity via ROS signaling.Collectively,these findings reveal a previously unidentified mechanism underlying the arms race between viruses and plants.