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Molecular detection of Phaeomoniella chlamydospora in grapevines
Estianne Retief, Ulrike Damm, Jan van Niekerk and Paul Fourie
Keywords: Black goo, Petri disease, Phaeomoniella chlamydospora, grapevine decline, trunk diseases
Introduction
There are several trunk diseases that cause a drastic decline in the productivity and health of grapevines. Dieback of young vines is most commonly ascribed to Petri disease (formerly known as Black goo). This disease can result in huge economic losses and is of great concern to most producers, viticulturists and researchers. The disease is caused by the fungus Phaeomoniella chlamydospora (Crous and Gams, 2000) and commonly occurs on young vines aged 1 to 5 years (Fourie et al., 2000). As the grapevine ages, the fungus will make the wood more susceptible to infection by other fungi and soft wood rotters in particular, such as Fomitiporia punctata. Esca disease ("beroerte") develops as the vine ages and typically occurs in vines aged 8 years and older, subsequently resulting in sudden dieback (Mugnai et al., 1999).
Not much is known about the epidemiology of Petri disease, but it has been proven that infected rootstock cuttings are one of the most important inoculum sources. The fungus presumably spreads from infected mother plants to rootstock cuttings (Crous et al., 1996; Edwards et al., 2003; Fourie and Halleen, 2002; 2003; Mugnai et al., 1999). Symptoms are generally not visible in one-year-old wood (Fourie et al., 2000), but in the past the fungus has often been isolated from apparently healthy propagation material. Stress conditions such as severe pruning, drought, poor drainage, nutrition deficiencies and soil compaction are prerequisites for symptom expression. (Ferreira, 1998; Ferreira et al., 1999). Typical symptoms of Petri disease include stunted growth, shorter internodes, small leaves, smaller trunks and branches and a general decline of young vines resulting in plant death (Bertelli et al., 1998; Ferreira, 1998; Fourie et al., 2000; Morton, 1995; Sidoti et al., 2000; Whiteman et al., 2003).
It has been found that the fungus is present in apparently healthy propagation material in a latent or endophytic form (Bertelli et al., 1998; Edwards and Pascoe, 2002) and it is therefore very difficult to trace the fungus. The most commonly used method to detect infection is by isolating the fungus onto an artificial growth medium. However, Pa. chlamydospora grows very slowly and it takes approximately 3 to 4 weeks from isolation to identification (Fig. 1). Other fungi that are isolated together with Pa. chlamydospora often surpass this fungus in growth. To speed up and facilitate diagnosis of the disease and research, there is consequently a large demand for a rapid, though sensitive and accurate detection method.
Serological techniques have been developed in the past for the identification of fungi and bacteria. DNA (deoxyribonucleic acid) "dot-blotting" techniques have been developed, in which specific hybridisation probes have been used. The above technique was not sufficiently sensitive, however, and was very time-consuming (Schaad and Frederick, 2002). Subsequently the PCR (polymerase chain reaction) technique was developed, providing the researcher with the desired sensitivity. A PCR is an in vitro method in which specific DNA sequences are synthesised and amplified by certain enzymes. Specific primers are required for initiation of the process and the two primers bind with the target DNA and cause amplification of the specific fragment. The amplified DNA undergoes gel-electrophoresis and depending on the "fingerprint" it is possible to determine whether the DNA of the specific fungus is present or not. The accuracy of the technique, however, depends on the specificity of the primers, which will only initiate PCR if the specific target DNA is present in the sample.
Primers that are Pa. chlamydospora specific (PCL1 and PCL2), have been developed by Groenewald et al. (2002). These researchers succeeded in detecting Pa. chlamydospora DNA in inoculated tissue culture plants using a PCR reaction. However, due to the presence of PCR inhibitors, this method will not be suited for molecular detection from lignified wood. Researchers in New Zealand (Ridgway et al., 2002) extracted DNA from grapevine wood using a commercial kit (Green and Thompson, 1999), where after they detected Pa. chlamydospora with a species-specific PCR. This technique was sufficiently sensitive to detect as little as 1 pg Pa. chlamydospora DNA (this is 10-12 g DNA or as few as 2 fungal spores). However, the commercial package is very expensive.
Further criticism of the use of PCR detection techniques in disease diagnosis, is that DNA of dead pathogen tissue can also be detected. Hot water treatment (50§C for 30 min.) of grapevine propagation material kills various pathogens in grapevine tissue (Goheen et al., 1973; Von Broembsen and Marias, 1978). A drastic decline in Pa. chlamydospora levels could be observed following hot water treatment (Fourie and Halleen, 2004). The question thus arises to which extent the hot water treatment will influence the sensitivity of the molecular detection technique.
The aim of this study was therefore to develop a cheap, though sensitive DNA extraction protocol for the detection of Pa. chlamydospora with a classical PCR reaction. A diagnostic technique of this nature can be used to diagnose grapevine diseases and phytosanitary certification of propagation material. The effect of hot water treatment on the sensitivity of this technique should therefore also be determined.
Material and Methods
For the development of the DNA extraction protocol grapevines that were naturally infected with Pa. chlamydospora were used. Various aspects of the above-mentioned DNA extraction protocols were adapted and combined in order to develop the ideal, though cost-effective protocol. Next the sensitivity of the protocol was determined and the accuracy thereof verified. To determine the sensitivity of the PCR reaction, known quantities of Pa. chlamydospora DNA (determined using a fluorometer) were added to a DNA solution, consisting of wood extractions that previously tested negative with the Pa. chlamydospora specific primers (Pch1 and Pch2) (Tegli et al., 2000).
For the verification of the protocol nursery plants were taken from various grapevine nurseries. The various rootstocks that were used, were 101-14 Mgt, Ramsey and 99 Richter in 3 different combinations (18 vines per bundle). To determine the occurrence of Pa. chlamydospora in the standard way (Fourie and Halleen, 2002, 2003, 2004), isolations were made 2-5 cm from the base of the rootstock on PDA-medium. This same segment was then used for molecular detection.
To determine the effect of hot water treatment on molecular detection, Chardonnay/101-14 Mgt nursery plants were used. The grapevines were divided into six bundles of eight grapevines each. Three of the bundles were drenched in hot water (50§C) for 30 min, while the other three bundles were left untreated as a control. The results of isolations and molecular detection of Pa. chlamydospora in the basis of rootstocks from the respective bundles were compared.
Results
PCR with the Pa. chlamydospora specific primers was successful in several of the samples that had been tested. The PCR technique was sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic DNA (Fig. 2). Pa. chlamydospora was detected in all the samples that indicated a positive result with the isolations (Fig. 3). The pathogen was moreover detected in various grapevines that tested negative with isolations, using the molecular technique.
Pa. chlamydospora was isolated from 90% of the non-hot water treated grapevines, but could not be isolated from any of the grapevines that had been treated with hot water. However, the molecular detection technique detected Pa. chlamydospora DNA in 100% of the hot water treated and non-hot water treated grapevines (Fig. 4). As could be expected, dead pathogen tissue could be detected with the molecular technique and hot water treatment did not influence the sensitivity of molecular detection. The fact that no fungi were isolated after the hot water treatment, once again emphasises the importance of this treatment for the proactive control of Petri disease in nurseries (Fourie and Halleen, 2004).
Discussion
The successful development of this molecular detection technique creates excellent opportunities for research on grapevine trunk diseases. Detection of the Petri disease pathogen, Pa. chlamydospora, in vine material may be accelerated from the current 3-4 weeks to 1-2 days. Cost estimates have shown, moreover, that this locally developed extraction protocol is up to 20 times cheaper than the imported kits that are available commercially.
If specific primers for other trunk pathogen, e.g. Eutypa lata ("tandpyn"), Botryosphaeria spp. ("swart-arm"), Phomopsis viticola ("streepvlek" and dead-arm) and Cylindorcarpon spp. ("swartvoet"), are developed, this detection technique will enable researchers to undertake cutting edge research on the inoculum sources, disease cycles, host-pathogen interactions, epidemiology and management of these diseases. This technique can moreover become an important aid in the certification of grapevine propagation material. Although hot water treatment considerably reduced the occurrence of Pa. chlamydospora in rootstock cuttings and uprooted nursery vines, it did not entirely kill the fungus (Fourie and Halleen, 2004). However, the dead pathogen tissue will still be detected by the molecular technique. Further molecular studies could focus on the effect of hot water treatment on RNA (ribonucleic acid) level. Theoretically, transcripts of RNA will have a very brief survival period after the pathogen has been killed. RNA detection is therefore only possible from live organisms (Klein and Juneja, 1997).
The molecular technique will shortly be used to investigate various stages in the nursery so as to detect possible inoculum sources of Pa. chlamydospora. Knowledge of the epidemiology of the pathogen is necessary to formulate and conduct research on disease management strategies.
Acknowledgement
This research was funded by Stellenbosch University, Winetech (Project US/PP 03/2001) and the Foundation for Research Development (GUN no. 2054222).
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