Detection of Huanglongbing (Citrus Greening) Disease by Nucleic Acid Spot Hybridization

Polymerase chain reaction (PCR) amplification with primers specific to the rDNA region successfully amplified the 1160-bp DNA fragment from a Huanglongbing (HLB)-infected sweet orange sample with mottling symptoms leaves, but not from healthy sweet orange plants. The PCR product of 1160-bp was used as probe labeled with biotin for detection of the HLB pathogen in the nucleic acid spot hybridization (NASH) test. It was found that the HLB pathogen could be detected up to 1:100 dilution in HLB-infected tissue. Total DNA extracted from HLB-infected tissue was diluted 2-fold as 900 ng in TE buffer and spotted on a nitrocellulose membrane. Strong signals were observed up to 225 ng of DNA dilution, whereas a moderate signal was recorded at 112 ng. No hybridization signal was observed in the healthy samples, while strong signals were observed in the positive control

Antiviral and Antiviroid Activity of MAP-Containing Extracts from Mirabilis jalapa Roots

Extracts of Mirabilis jalapa (Nyctaginaceae), containing a ribosome inactivating protein (RIP) called Mirabilis antiviral protein (MAP), were tested against infection by potato virus X, potato virus Y, potato leaf roll virus, and potato spindle tuber viroid. Root extracts of M. jalapa sprayed on test plants 24 h before virus or viroid inoculation inhibited infection by almost 100%, as corroborated by infectivity assays and the nucleic acid spot hybridization test. Antiviral activity of MAP extracts was observed against mechanically transmitted viruses but not against aphid-transmitted viruses. Purified MAP showed the same antiviral effect as the crude extracts.

Development of the molecular methods for potato virus and viroid detection and prevention

Potato is the fourth most important food crop in the world and it forms the diet of a billion consumers in developing countries, where potato production is increasing rapidly. However, potato virus diseases in developing countries are one of the major causes of lower yields. Their control requires the development of appropriate virus-detection and seed-production technologies for the region. Recent progress in developing nucleic acid based virus detection methods are reviewed. Refinements of the protocols applicable to the laboratories located in seed producing areas are discussed. Nucleic acid spot hybridization (NASH) and reverse transcription polymerase chain reaction (RT-PCR) methods are described for the detection of viruses and viroids in dormant seed tubers and insect vectors. Although the potato crop is susceptible to over 25 virus and viroid diseases, only universally economically important viruses have been dealt with here. The progress of pathogen-derived resistance for the control of potato virus diseases is elaborated, and the results of field tests indicate their feasibility in virus control.Key words: dot-blot, spot-hybridization, reverse transcription, polymerase chain reaction, transgenic plants.