Abstract
BackgroundGremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed.ResultsWe designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background.ConclusionThe methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities.
Highlights
Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species
None of the 14 conifer species produced any amplicon with this primer pair, demonstrating the incompatibility of the primers to conifer 18S ribosomal DNA (rDNA)
The specific primers developed in this study successfully detected G. abietina at species level and, can function as rapid molecular markers for its identification and detection in composite fungal or plant samples without the need for isolation and cultivation. By verifying these DNA markers in a wide range of conifer species, the present study indicates that this detection system can be applied to all potential hosts, so it should be a valuable forest management tool across broad geographic regions
Summary
Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. Gremmeniella abietina (Lagerb.) Morelet is among the most destructive conifer forest pathogens in the northern hemisphere. This ascomycete fungus has a broad host range and causes stem canker and shoot dieback in many conifer species of the genera Pinus, Abies, Picea, Larix, Tsuga and Pseudotsuga [1,2,3]. The fungus may grow in the host as an endophyte for more than one year [7,8], and infected trees can remain undetected for several years before manifesting visible symptoms This poses difficulties for diagnosing disease at an early stage, using asymptomatic materials. Physiological, pathogenic and biochemical characters and (page number not for citation purposes)
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