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Molecular detection of potential inoculum sources of Phaeomoniella chlamydospora in grapevine nurseries
Estianne Retief, Adéle McLeod & Paul Fourie, Plant Pathology Department, Stellenbosch University
Key words: Black goo, Petri disease.
Introduction
Petri disease (previously known as "Black goo") is a common occurrence on grapevines aged 1 to 5 years (Fourie et al., 2000). The disease reduces the chances of survival of young grapevines (Mugnai et al., 1999). External symptoms of Petri disease are stunted growth, shortened internodes, small leaves, and a general deterioration of young vines which may result in the plant’s dying (Morton, 1995; Bertelli et al., 1998; Ferreira, 1998; Fourie et al., 2000; Sidoti et al., 2000; Whiteman et al., 2003). Internal symptoms include a black discolouration of the xylem vessels, as well as tyloses formation (balloon-like growths) in these vessels (Ferreira, 1998). Phaeomoniella chlamydospora is considered to be the main causal organism (Fourie and Halleen, 2001; Wallace et al., 2003; Whiteman et al., 2003).
Not much is known about the epidemiology of P. chlamydospora, although a few sources of inoculum have been identified. Spores of P. chlamydospora have been captured in vineyards in California and France (Larignon, 1998; Eskalen et al., 2003). From these it was possible to deduce that the fungus produces spores (conidia) that are distributed aerially and penetrate the grapevine mainly through pruning wounds (Larignon, 1998; Eskalen et al., 2003).
Infected rootstock mother plants and propagation material are considered to beprimary inoculum sources, as isolations have shown the pathogen to be present in apparently healthy rootstock mother plants (Fourie and Halleen, 2004a) and cuttings (Bertelli et al.,1998; Larignon, 1998; Fourie and Halleen, 2002; Halleen et al., 2003). Spores and/or hyphal fragments of P. chlamydospora have been found in the entire length of rootstock canes (Feliciano en Gubler, 2001; Edwards et al., 2003) and one wonders whether the spores might be transported to the canes in the juice flow of infected rootstock mother plants (Edwards et al., 2003).
Infested soil is also considered a potential source of inoculum, as P. chlamydospora has been detected in nursery and vineyard soil by means of a conventional species- specific PCR (polymerase chain reaction) (Damm and Fourie, 2005). If P. chlamydospora forms hardy spores (chlamydospores), the fungus may survive in the soil for long periods. One expects chlamydospores to form conidia, which will then infect damaged roots of grapevines in nurseries and grapevines (Bertelli et al., 1998; Feliciano and Gubler, 2001).
It is difficult to detect P. chlamydospora using traditional isolation methods, due to the slow growth of the fungus and a lack of selective media. For this reason a conventional PCR technique was devised to detect P. chlamydospora in grapevine wood (Retief et al., 2005). The technique is sufficiently sensitive to detect 1 pg P. chlamydospora DNA (deoxyribonucleic acid) in grapevine wood, but not sufficiently sensitive to detect the DNA of the fungus in soil and water. A similar technique has been used in New Zealand and P. chlamydospora was detected during all the nursery stages - especially in drench solutions, where there was repeated exposure to plant material, e.g. in hydration tanks. The percentage of positive samples was lower for grafting equipment and callusing media (Whiteman et al., 2003).
The first objective of this study was to finetune DNA extraction and conventional PCR techniques that had previously been developed and optimised for grapevine wood and soil (Damm and Fourie, 2005; Retief et al., 2005), for molecular detection in water and callusing medium.
The subsequent objective of the study was to use these molecular techniques to determine whether P. chlamydospora was present in samples (water, soil, graft wood and callusing medium), collected from different nurseries and at different stages, in order to identify the different inoculum sources of the fungus during the various nursery stages in South Africa.
Material and methods
Sampling: Rootstock canes of the cultivars 101-14 Mgt, Ramsey, 99 Richter and 110 Richter were collected from the rootstock mother plants at six nurseries. Rootstock cuttings (101-14 Mgt and Ramsey) and scion cuttings (various cultivars) were also collected from 16 and 19 different nurseries respectively during the grafting process.
Soil samples were taken from the root zone of the mother plants from which the rootstock canes had been collected. Soil samples were also taken from the prepared nursery beds at 18 nurseries just before planting out occurred.
During the first hydration (before cold storage) two water samples each were collected from 15 different nurseries. The one sample was water from the hydration tank in which rootstock cuttings had been drenched for a period of 12 hours. The other sample was taken from the water source (that supplies water to the hydration tank), in order to determine whether the water source was contaminated. Water samples were also taken from 21 nurseries during the second hydration, during the grafting process (before grafting or just before callusing).
During the grafting process callusing medium was collected from 12 nurseries. The samples were taken before the callusing period, in other words before the grafted cuttings were packed in the callusing medium and before the latter was drenched in a fungicide suspension.
Molecular detection: DNA was extracted from the soil and wood using the extraction protocols that had already been developed (Damm and Fourie, 2005; Retief et al., 2005). Different aspects of the latter protocols were used to design new protocols for DNA extraction from water and callusing medium. For the DNA extraction from water 90 ml of water was centrifuged in a centrifuge to collect all material at the bottom of the tube. The remaining fluid was discarded, and a CTAB extraction buffer added to the material to form a suspension. Thereafter glass balls were added to the suspension, and it was shaken to break the cell walls for DNA extraction. Subsequently the same protocol as for DNA extraction from wood was followed. DNA extraction from the callusing medium took place firstly by breaking the cells. The cells were broken by rapidly shaking the callusing medium and glass balls that had been frozen with liquid nitrogen. A CTAB extraction buffer was then added and the same protocol as for DNA extraction from wood was followed.
A one-tube nested-PCR technique (Olmos et al., 1999) was then used in combination with species-specific primers to detect P. chlamydospora in water, soil, graft wood and callusing medium. With a nested-PCR all fungal DNA is firstly multiplied from the sample, whereafter a PCR is done with the species-specific primers Pch1 and Pch2 (Tegli et al., 2000) for P. chlamydospora DNA. By doing the entire process in a single tube, the chances of contamination are drastically reduced.
The sensitivity of the PCR reaction was determined by adding known quantities of P. chlamydospora DNA (as determined with a fluorometer) to a DNA solution, consisting of DNA extractions from wood, soil, water and callusing medium, which had tested negatively with the P. chlamydospora specific primers (Pch1 and Pch2) in the past. To determine how many spores may be detected in water samples, spore suspensions of P. chlamydospora were added to water samples to obtain final concentrations of 101 and 102 conidia in 90 ml of water, whereafter the DNA was extracted from these samples.
The identity of PCR products obtained, using P. chlamydospora specific primers, was confirmed by restriction enzyme analysis and DNA sequencing.
Results
The nested-PCR was sensitive enough to detect 1 fg P. chlamydospora genomic DNA from water and 10 fg from the callusing medium, wood and soil. It was also possible to detect as few as 10 spores in a water sample of 90 ml (Fig. 1).
Samples that tested positively for P. chlamydospora were rootstock canes collected from mother plants (25%), rootstock cuttings collected during grafting (42%), scion cuttings collected during grafting (16%), water samples collected during the first hydration before cold storage (40%), water samples collected during grafting (67%), callusing medium (8%) and soil samples collected from rootstock mother blocks (17%) (Fig. 2). However, P. chlamydospora was not detected in soil samples from nursery beds before planting. Various PCR products were obtained using the one-tube nested-PCR, whereafter it was confirmed by means of restriction enzyme analysis and/or DNS sequence determination that the products were indeed P. chlamydospora. P. chlamydospora in mother blocks was detected mostly in 101-14 Mgt (13%) and 99 Richter (8%), as opposed to Ramsey (4%) and 110 Richter (0%). During grafting P. chlamydospora was also detected mostly in 101-14 Mgt (24%) as opposed to Ramsey (18%).
Discussion
In support of previous studies, it was once again confirmed that infected mother plants and cuttings are the primary inoculum source, in that a quarter of the rootstock canes from mother plants tested positively for the presence of P. chlamydospora. The rootstock canes 101-14 Mgt had the highest percentage of infected canes which correlates with the findings of Fourie and Halleen (2004a), who used conventional isolation techniques.
Infested soil should also be considered a potential inoculum source, as P. chlamydospora DNA was also detected in soil samples taken from mother blocks. P. chlamydospora may be present in the soil as mycelium, conidia, chlamydospores and/or other fruit structures, deriving from infected mother plants. The long term survival of this fungus in the soil is nevertheless questioned seeing that it could not be detected in soil samples from nursery beds after a one year period of lying fallow. Another possible explanation for this absence is the levels of the pathogen being so low as to make it impossible to detect through PCR.
Water samples taken before cold storage, during grafting and from fungicide tanks, also tested positively for the presence of P. chlamydospora. None of the water samples taken from the water sources tested positively for the pathogen, which proves that the water source is not a source of inoculum. After a hydration period of 12 hours the water was contaminated, however, most probably due to contaminated cuttings. The contaminated hydration water is therefore a very important source of inoculum, seeing that all cuttings undergo the hydration process before cold storage. Similar results were also obtained in New Zealand (Whiteman et al., 2003). Sanitation practices, as recommended by Fourie and Halleen (2006a,b), are therefore of the utmost importance in this regard.
From the results with rootstock cuttings it seems that the number of infected samples increase during the nursery process, from 25% in mother plants to 42% during grafting. The same material was not used in these instances, however, and further samples will have to be analysed to confirm this preliminary conclusion. Scion cuttings (16%) also tested positive for P. chlamydospora during grafting. Just like rootstock cuttings, these cuttings may be infected during hydration or possibly from infected mother plants.
A very small number of the callusing medium samples (8%) tested positive for P. chlamydospora. Callusing medium consists mainly of pine sawdust and it is not known whether P. chlamydospora occurs on pines. This might indicate that the callusing medium was contaminated during the nursery stages, most likely from contaminated water, utensils or floors.
Several potential inoculum sources were identified during the study. By applying the correct control measures, the inoculum may be reduced or even killed. Up to now warm water treatment (30 min at 50°C) of propagation material before grafting has been proven most effective in reducing P. chlamydospora levels in naturally infected rootstock cuttings (or dormant nursery plants) (Fourie and Halleen, 2004b).
Hydration tanks are also an important focus of control strategies. Hydration tanks must be sterilised after each hydration and the water in which cuttings are immersed must be treated with chemical and/or biological control agents, since uncovered pruning wounds on cuttings provide the perfect point of entry for P. chlamydospora (Messina, 1999; Fourie et al., 2001; Fourie and Halleen, 2004b). South African nurseries use broad spectrum contact fungicides such as chinosol, captan and/or iprodione in hydration tanks, but these fungicides were shown to be moderately or poorly effective at reducing in vitro germination and mycelium growth of P. chlamydospora (Groenewald et al., 2000; Jaspers, 2001). Recent semi-commercial nursery trials by Fourie and Halleen (2006a, b) indicated that the use of benomyl (100 g/100 L water) or Sporekill (150 mL/100 L water) in hydration and drench water during all stages of preparation and grafting of propagation material, resulted in the biggest reduction in infection levels of P. chlamydospora.
The detection of P. chlamydospora in this study was based on the presence of the pathogen’s genomic DNA. It is therefore important to take into account that the presence of the pathogen’s DNA does not confirm the pathogen to be viable. Further studies will therefore have to focus on the detection of RNA (ribonucleic acid). Theoretically transcripts of RNA will have a very short survival period once the pathogen has been killed. RNA detection is therefore only possible from live organisms (Klein and Juneja, 1997).
Acknowledgement
This research was funded by Stellenbosch University, Winetech (Project US/PP 03/2001) and the Foundation for Research Development (GUN no. 2054222).
Literature
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Eskalen, A., Rooney-Latham, S.N., Feliciano, A.J. and Gubler, W.D., 2003. Epiphytic occurrence of esca and Petri disease pathogens on grapevine tissues. In 3rd International Workshop on Grapevine Trunk Diseases, Lincoln, New Zealand, pp. 29.
Feliciano, A.J. and Gubler, W.D., 2001. Histological investigations on infection of grape roots and shoots by Phaeoacremonium spp. Phytopathologia Mediterranea 40, S387 - S393.
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Groenewald, M., Denman, S. and Crous, P.W., 2000. Fungicide sensitivity of Phaeomoniella chlamydospora, the causal organism of Petri grapevine decline. South African Journal of Enology and Viticulture 21, 59 - 61.
Halleen, F., Crous, P.W. and Petrini O., 2003. Fungi associated with healthy grapevine cuttings in nurseries, with special reference to pathogens involved in the decline of young vines. Australasian Plant Pathology 32, 47 - 52.
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Retief, E., Damm, U., van Niekerk, J.M., McLeod, A. and Fourie, P.H., 2005. A protocol for molecular detection of Phaeomoniella chlamydospora in grapevine wood. South African Journal of Science 101, 139 - 142.
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Summary
Petri disease of grapevine is primarily caused by Phaeomoniella chlamydospora. This pathogen affects mostly young grapevines, but is also implicated in esca disease of older grapevines. Little is known about the disease cycle of this fungus. Infected propagation material was identified as a major means of dissemination of the pathogen. Recently, the pathogen was also detected from soil in South Africa and airborne conidia have been found in vineyards. The aim of this study was to use a molecular detection technique to test different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from nurseries in South Africa at different nursery stages for the presence of P. chlamydospora. A one-tube nested-PCR technique was optimised for detecting P. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. The one-tube nested-PCR was sensitive enough to detect as little as 1fg of P. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. Altogether the molecular detection technique revealed the presence of P. chlamydospora in 35% of rootstock cuttings, 16% of scion cuttings, 58% of hydration water, 17% of soil and 8% of callusing medium samples. These media can therefore be considered as possible inoculum sources of the pathogen during the nursery stages.
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