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 techniq...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.展开更多
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 easil...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.展开更多
基金supported by the National Natural Science Foundation of China(31272015)the National Basic Research Program(973)of China(2014CB138400)the Special Fund for Agro-scientific Research in the Public Interest,China(201303021,201303028)
文摘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.
文摘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.
