Use of next‐generation sequencing for the identification and characterization of Maize chlorotic mottle virus and Sugarcane mosaic virus causing maize lethal necrosis in Kenya

Abstract

The diagnosis of novel unidentified viral plant diseases can be problematic, as the conventional methods such as real‐time PCR or ELISA may be too specific to a particular species or even strain of a virus, whilst alternatives such as electron microscopy (EM) or sap inoculation of indicator species do not usually give species level diagnosis. Next‐generation sequencing (NGS) offers an alternative solution where sequence is generated in a non‐specific fashion and identification is based on similarity searching against GenBank. The conventional and NGS techniques were applied to a damaging and apparently new disease of maize, which was first identified in Kenya in 2011. ELISA and TEM provided negative results, whilst inoculation of other cereal species identified the presence of an unidentified sap transmissible virus. RNA was purified from material showing symptoms and sequenced using a Roche 454 GS‐FLX+. Database searching of the resulting sequence identified the presence of Maize chlorotic mottle virus and Sugarcane mosaic virus, a combination previously reported to cause maize lethal necrosis disease. Over 90% of both viral genome sequences were obtained, allowing strain characterization and the development of specific real‐time PCR assays which were used to confirm the presence of the virus in material with symptoms from six different fields in two different regions of Kenya. The availability of these assays should aid the assessment of the disease and may be used for routine diagnosis. The work shows that next‐generation sequencing is a valuable investigational technique for rapidly identifying potential disease‐causing agents such as viruses.

Production of a Polyclonal Antibody against the Recombinant Coat Protein of the Sugarcane Mosaic Virus and Its Application in the Immunodiagnostic of Sugarcane

“…The use of next-generation sequencing (NGS) for the identification and characterization of maize and sugarcane viruses has been reported [25,26]. It was claimed that the NGS method provides a powerful and rapid identifying potential diseases caused by virus, compared to the molecular and serological techniques.…”

Insights of maize lethal necrotic disease: A major constraint to maize production in East Africa

“…(Mahuku et al, 2015a, b). In spite of serological methods such as ELISA being less accurate in identifying unusual or variant isolates because of being too specific to a particular species or even strain of a virus as reported by Adams et al (2013), still it can be used in identification because it is the easiest method associated with low cost. Furthermore, it is rapid and can be used in the identification of large number of samples and that is why it is intensively used in quarantine/movement of seeds and plants across countries to identify diseases of quarantine importance including MLND (Mezzalama et al, 2015).…”

“…ELISA method have been used to identify WSMV in wheat (Montana et al, 1996;Ilbagi et al, 2005), MCMV in maize (Jensen et al, 1991;Xie et al, 2011;Adams et al, 2013;Lukanda et al, 2014), SCMV in maize (Louie, 1980;Adams et al, 2013;Lukanda et al, 2014) and MDMV in maize (McDaniel and Gordon, 1985;Giolitti et al, 2005). DAS-ELISA has been used to identify Kiruwa et al 273 MLND causing viruses in Kenya but gave negative results (Adams et al, 2013) probably due to low sensitivity and poor specificity for unusual or variant isolates (Adams et al, 2013). Similar study was done to identify MCMV and SCMV by ELISA (DAS-ELISA and Indirect ELISA) with polyclonal antibodies produced against the East African strains of MCMV and SCMV and it was successful.…”

“…The best method of controlling plant diseases is proper identification of the causative agents (Webster et al, 2004;Adams et al, 2013) and this is supported by the best diagnostic tools. Several methods have been used to diagnose plant viral diseases.…”

“…In 2011, a disease with virus like symptoms (chlorotic mottle on maize leaves, mild to severe mottling and necrosis) were reported in east Africa causing dramatic maize damage in farmers fields (Wangai et al, 2012a,b). The disease was identified as Maize Lethal Necrotic Disease (MLND) (Wangai et al, 2012b;Adams et al, 2013), a new disease in Africa and perhaps the worst enemy of the maize crops in recent times. This review discusses MLND in east Africa, including its importance, diagnostics, etiology, managements and therefore highlights the future research needs.…”