Author
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Lupiani, Blanca |
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Raina, Ashok |
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Huber, Curtis |
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Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 7/28/1999 Publication Date: N/A Citation: N/A Interpretive Summary: The corn earworm, Helicoverpa zea, is a major pest affecting a wide variety of crops such as corn, tomato, soybean and cotton. The primary control strategy for this pest is the use of chemical pesticides. However, in the last few years, increasing efforts have been directed toward the use of microbial agents to control this pest. An example of a potential biocontrol agent is the Helicoverpa zea reproductive virus (HzRV). HzRV infects corn earworm making the adult moths sterile. A bioassay was previously developed to detect the presence of this virus in infected insects. However, this bioassay is time consuming and requires more than a month to complete. In the present study we have developed a polymerase chain reaction (PCR) assay that allows the detection of minute amounts of viral DNA in tissue samples. The assay was shown to be highly specific, sensitive and fast taking less than one day. This PCR assay will be useful for other scientists interested in studying the incidence of the virus in wild populations of H. zea as well as its tissue and host specificity. These are basic biological properties of the virus that have to be addressed before the virus can be used as a biocontrol agent. Technical Abstract: Helicoverpa zea reproductive virus (HzRV) is a non-occluded bacilliform virus that affects both female and male moths of the corn earworm, Helicoverpa zea. In order to study the biology and host range of HzRV, previously a bioassay was developed to detect the presence of this virus in infected insects. A draw back of this bioassay is that it is time consuming gand requires more than a month to complete. Here we describe the development of a polymerase chain reaction (PCR) assay for the rapid detection of HzRV in infected corn earworms. The genome of HzRV was was digested with Pst I and cloned into a plasmid vector. Sequences from two different clones, P4 and P13 were selected for designing two sets of primers. These primers were used for PCR and their sensitivity and specificity to detect HzRV DNA were examined. Both sets of primers produced the expected amplification product in samples containing HzRV DNA but not in uninfected corn earworm samples, Spodoptera frugiperda (Sf-9) cells or samples from the Autographa californica nuclear polyhedrosis virus (AcNPV). In addition, the primer pair of the clone P13, was sensitive enough to detect approximately 175 copies of viral DNA. We then used this assay to examine feral populations of H.zea from seven geographical locations in the U.S. HzRV was detected primarily in the Mississippi populations and to a lesser extent in Iowa and Georgia, but none in Maryland, Missouri and Texas populations. This PCR assay provides a highly specific, sensitive and rapid way of detecting the presence of HzRV and will be useful in further studying the host range, tissue specificity and incidence of this virus in wild populations of the corn earworm. |
