Molecular detection of Phaeomoniella chlamydospora in grapevine nurseries

[摘要] ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causessevere decline and dieback of young grapevines (1-7 years old) and also predisposes the woodfor infection by other pathogens. Knowledge about the epidemiology and especially inoculumsources of this disease is imperative for subsequent development of management strategies.Through isolation studies it was shown that Pa. chlamydospora is mainly distributed throughinfected propagation material in South Africa. However, the infection pathways and inoculumsources in grapevine nurseries are still unclear. The only existing method to detect this pathogenin various media is by means of isolation onto artificial growth media. This has proven to beproblematic since this fungus is extremely slow growing (up to 4 weeks from isolation toidentification) and its cultures are often over-grown by co-isolated fungi and bacteria before itcan be identified. The aim of this study was (i) to develop a protocol for the molecular detectionof Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to testdifferent samples (water, soil, rootstock and scion cuttings and callusing medium) collected fromnurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora.A protocol was developed and validated for the molecular detection of Pa.chlamydospora in grapevine wood. Firstly, several previously published protocols were used todevelop a cost-effective and time-efficient DNA extraction method from rootstock pieces ofpotted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 andPch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomicDNA from grapevine wood. The protocol was validated using various grapevine material from 3different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3different nurseries, including grapevines that were subjected to hot water treatment. The basalend of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificialmedium and molecular detection. The identity of PCR products obtained from a subset ofsamples, that only tested positive for Pa. chlamydospora based on molecular detection, wasconfirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Moleculardetection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, themolecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1%of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydosporawas not isolated from hot water treated samples. The results confirm the importance of hot watertreatment for proactive management of Petri disease in grapevine nurseries. However, Pa.chlamydospora DNA was molecularly detected in hot water treated samples in frequenciessimilar to that detected in non-hot water treated samples. As expected, the DNA in hot watertreated plants was not destroyed and could be detected by the developed molecular detectionprotocol. This is an important consideration when using molecular detection for diseasediagnosis or pathogen detection and shows that these methods should be used in conjunctionwith other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10to 15 times cheaper than commercial DNA extraction kits.Preliminary studies showed that the aforementioned molecular detection technique wasnot specific and sensitive enough for detection of Pa. chlamydospora in soil and water(unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa.chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood.Rootstock cane sections and soil samples were taking from the mother blocks from severalnurseries. Water samples were collected from hydration and fungicide tanks during pre-storageand grafting. Scion and rootstock cuttings were also collected during grafting and soil werecollected from the nursery beds prior to planting. The one-tube nested-PCR was sensitiveenough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg fromwood, callusing medium and soil. PCR analyses of the different nursery samples revealed thepresence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequenceanalyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bandswere Pa. chlamydospora specific, except for five bands obtained from callusing media and oneband from water. Considering only Pa. chlamydospora specific PCR bands, the moleculardetection technique revealed the presence of Pa. chlamydospora in 25% of rootstock canesections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttingscollected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12-hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of thecallusing medium samples. These media should therefore be considered as potential inoculumsources or infection points of the pathogen during the nursery stages. The results furthermoreconfirmed previous findings that Pa. chlamydospora is mainly distributed through infectedrootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock motherplants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological orchemical amendments in the hydration water and callusing medium and wound protection fromsoil borne infections.

[发布机构] Stellenbosch University