A new perspective on soilborne diseases of grapevines in nurseries

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A new perspective on soilborne diseases of grapevines in nurseries

Gert van Coller

by G.J. van Coller^A,B,F S. Denman^A,C
S.C. Lamprecht^A,E and P.W. Crous^A,D
A. Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Matieland, 7602.
B. Current address: Department of Agriculture: Western Cape, Private Bag X1, Elsenburg 7607.
C. Forest Research Station, Alice Holt Lodge, Farnham, Surrey, GU10 4LH, England.
D. Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
E. ARC-PPRI, Vredenburg, Stellenbosch, 7599, South Africa.
F. Corresponding author; email: gertvc@elsenburg.com
Keywords: Grapevine nursery, soilborne diseases
Abstract
Research on soilborne diseases of nursery grapevines in the Western Cape Province in the 1970’s demonstrated that Phytophthora cinnamomi was the most important fungal pathogen. With the recent increase in vineyard plantings and only moderately successful propagation of nursery stock, a re-assessment of soilborne fungal pathogens associated with diseased grapevines in nurseries was undertaken. Declining plants of the most popular rootstock/scion combinations were collected from three commercial nurseries at Wellington between February and April 2001 (survey one), November and December 2001 (survey two), and March and April 2002 (survey three). Isolations were made from root tissue. Potentially pathogenic fungi including Cylindrocarpon, Fusarium, Pythium and Rhizoctonia spp. were consistently isolated from roots. Phytophthora cinnamomi was infrequently isolated in the first survey, and not obtained at all during the second or third surveys. These results suggest that the status of soilborne fungal pathogens has changed considerably over the last 30 years. Since only three nurseries were surveyed, it is of the utmost importance that further surveys be conducted in representative areas of grapevine production.
What is known?
Soilborne diseases of grapevines pose a complex problem and limited information is available on these diseases, both locally and internationally, although sustainable production is affected. Previously Phytophthora and Pythium were considered the most important soilborne pathogens of grapevines, both in nurseries and vineyards (Chiarappa, 1959; Bumbieris, 1972; Mansilla, Pintos & Salinero, 1993). However, other pathogens such as Cylindrocarpon spp. (Grasso, 1975), Fusarium spp. (Andrade, 1993; Grasso, 1984; Highet & Nair, 1995; Lele et al., 1978), Rhizoctonia solani (Walker, 1992, 1994, 1997) and Rosellinia necatrix (Veghelyi, 1991) also had a significant effect on plant health.

FIG. 1. Diseased patch in a grapevine nursery.

In South Africa, an extensive investigation regarding soilborne diseases of grapevines in nurseries and vineyards in the Western Cape Province was carried out during the 1970s by Dr P.G. Marais (Marais, 1979, 1980). Phytophthora cinnamomi was considered the main root pathogen but root rot caused by Pythium ultimum was also significant (Marais, 1979, 1980). Macrophomina phaseolina, R. solani, R. necatrix and Sclerotium rolfsii were less important (Marais, 1979, 1980; Keyser & Ferreira, 1988). Cylindrocarpon spp. are also important pathogens of grapevines, causing black foot rot disease (Fourie, Halleen & Volkmann, 2000; Fourie & Halleen, 2001).
The South African grapevine industry has expanded considerably and new cultivation practices, including the extensive use of new agricultural chemicals, are being used. Less than 50% of propagation material yields healthy saleable plants (P.H. Fourie, personal communication), and soilborne pathogens contribute significantly to the loss of propagation material. The aim of this research was to determine potentially pathogenic fungal genera associated with selected nursery grapevines. Information obtained from this study will be a first step towards developing sustainable disease management strategies for the nursery industry, with resulting financial benefits for the grapevine industry.
Materials and methods
Three surveys were conducted at three field nurseries (Fig. 1) at Wellington. Survey one was done between February and April 2001, survey two from November-December 2001, and survey three from March-April 2002. During the first and third surveys, grapevines were collected 5-7 months after planting, and 1-2 months after planting during the second survey. Plants with chlorotic leaves, stunted growth and dieback were sampled from two rootstocks, 99 Richter and 101-14 Mgt.

FIG. 2. Diseased roots of a nursery grapevine.

Roots of half the plants were surface disinfested, but the others were washed with tap water only, to rid roots of adhering soil particles. Diseased segments from roots (Fig. 2) were plated onto a range of culture media. Fungal colonies that developed were identified to generic level using the keys of Barnett and Hunter (1999). The percentage incidence of the various fungal genera was calculated by dividing the number of tissue pieces yielding a specific fungus by the total number of tissue pieces that were plated out and by then multiplying that number with 100.
Results
Fungal genera occurring in vines.

Nine fungal genera potentially pathogenic to grapevine roots and crowns were obtained, namely Botryosphaeria, Cylindrocarpon, Fusarium, Macrophomina, Phomopsis, Phytophthora, Pythium, Rhizoctonia, and Sclerotium.
Distribution of fungal genera at the different nurseries (Fig. 3).

Nursery 1 had a significantly higher incidence of Botryosphaeria than nurseries 2 and 3.
There was a higher incidence of Cylindrocarpon in nursery 1 than nursery 3, but a lower incidence of Rhizoctonia in nursery 1 than nursery 3.
It is interesting to compare the incidence of Rhizoctonia and Cylindrocarpon at nursery 1 and 3, where results suggest an inverse relationship between these two pathogens. Thus, where there is a high incidence of Rhizoctonia, a lower incidence of Cylindrocarpon occurs, and a high incidence of Cylindrocarpon is associated with a low incidence of Rhizoctonia.
At nursery 2 Sclerotium was present but it was not present at nurseries 1 or 3.
Nursery 2 also had a higher incidence of Fusarium and Macrophomina than nursery 3.
Fusarium was the most frequently isolated fungus followed by Cylindrocarpon and Rhizoctonia.
Surveys/Seasons (Fig. 4)

The incidence of Fusarium was highest during the late summer surveys (Surveys 1 and 3) but low in the spring (Survey 2).
On the other hand, the incidence of Pythium was low in the late summer season but higher in the Spring.
The incidence of Cylindrocarpon and Rhizoctonia was low in the second late summer sampling (Survey 3).
Phytophthora was present in late summer in the first year only.
Rootstocks (Fig. 5)

The only differences between rootstocks with regard to the incidence of pathogens was demonstrated by Pythium and Rhizoctonia, and an inverse relationship existed. Thus, 99 Richter supported a higher incidence of Pythium than 101-14 Mgt, but 101-14 Mgt had a higher incidence of Rhizoctonia than 99 Richter.
Discussion
A variety of potentially pathogenic fungal genera, including Cylindrocarpon, Fusarium, Pythium and Rhizoctonia were frequently obtained from declining grapevines. All of these genera are known pathogens on a wide variety of crops.
Certain species of Cylindrocarpon cause black foot rot (Maluta & Larignon, 1991), a disease common to grapevines in the Western Cape Province (Fourie et al., 2000; Fourie & Halleen, 2001). Halleen, Crous & Petrini (2003) demonstrated that Cylindrocarpon was present in less than 1% of plants prior to planting in nurseries (October), whereas 50% or more of the plants were infected at the end of the season (June). This clearly shows the importance of nursery soils as a source of inoculum for this disease and demonstrates that many nursery grapevines are already infected when planted into commercial vineyards.
In this study, Fusarium was frequently isolated from roots of diseased nursery grapevines and 10 species were identified. The pathogenicity of 3 commonly occurring species, viz. Fusarium oxysporum, F. proliferatum and F. solani was tested on grapevines grafted onto 99 Richter and 101-14 Mgt. Results indicated that all 3 species of Fusarium are pathogenic to both rootstocks. Fusarium oxysporum and F. solani are known as pathogens of grapevines in other countries, where F. oxysporum causes both root rot and wilt, and F. solani causes root rot only (Andrade, 1993; Grasso, 1984; Gugino, Travis & Stewart, 2001; Highet & Nair, 1995; Lele et al., 1978).
Species of Pythium comprised 36.4 % of isolates obtained by Marais (1980) during an extensive survey. They were identified, together with Phytophthora spp., as the most important soilborne pathogens of grapevines in nurseries in the Western Cape, with P. ultimum as the most important Pythium species. Considerably fewer isolates of Pythium were obtained during this survey, indicating that a possible shift in the status of soilborne pathogens may have occurred. This will have important ramifications in the development of disease management practices. Rhizoctonia was frequently isolated in this study, and was previously identified by Marais (1979) as a pathogen of the rootstock V. champini var. Salt Creek. However, Marais (1980) did not obtain Rhizoctonia from grapevine nurseries, but from mature vineyard plants. Halleen et al. (2003) also obtained isolates of R. solani from nursery grapevines in the Western Cape Province. However, no Rhizoctonia was detected in plants before they were transplanted in the field nurseries, but after three months in the nursery, Rhizoctonia was already present in a substantial number of nursery grapevines. Nursery soils can thus be seen as a primary source of inoculum for this pathogen. In spite of the obvious abundance of Rhizoctonia in grapevine plants and soils, and clear indications of its pathogenicity, much work remains to be done before effective management strategies can be devised. Rhizoctonia is a basidiomycete fungus with a complex life cycle. Nothing is known about how this life cycle is carried out on grapevines and what the epidemiology of the disease is. Rhizoctonia spp. also employ certain mechanisms such as anastomosis for the exchange of genetic material that provides these pathogens with the capacity to adapt to new environments and hosts, thereby potentially resulting in increased virulence and survival abilities. As a fundamental step towards understanding the role these organisms play in reducing production of grapevines it is essential to determine all the species of Rhizoctonia and the anastomosis groups involved. Clearly from both the results of this survey and previous studies (Walker, 1992, 1994, 1997), this needs to be addressed as a matter of urgency.
Certain pathogens appear to be absent from certain nurseries, e.g. Sclerotium that was only obtained from nursery 2, while the incidence of pathogens differs between nurseries. The highest levels of Rhizoctonia and the lowest levels of Cylindrocarpon was obtained from nursery 3, indicating a possible inverse relationship. However, more comprehensive surveys need to be conducted before these conclusions can be made with certainty.
In our surveys, Fusarium species were also obtained at significantly lower frequencies from roots during the second survey than during the first and third surveys, indicating that the level of infection of nursery grapevines with Fusarium increases as the season progresses. Similar results were obtained by Halleen et al. (2003), who also found an increase in the incidence of Fusarium spp. later in the season. The increased incidence is probably due to the fact that Fusarium spp. are soilborne organisms that can infect roots and multiply in a very short time, leading to higher levels of inoculum and consequent root disease. These results also suggest that Fusarium levels need to be reduced early in the season to prevent a high occurrence later in the season. Management strategies therefore need to address reducing Fusarium populations in both plant material and soil.
The levels of Pythium spp. seem to decline as the season progresses. This indicates the need to reduce levels of Pythium early in the season when the damage these pathogens cause would be maximised. These results indicate seasonal changes in the incidence and occurrence of different pathogens, and this is an epidemiological aspect that requires further investigation. An unexpected result of the survey was that Phytophthora was rarely obtained, and only from the rootstock 99 Richter. Since Phytophthora and Pythium were previously deemed the most important and devastating fungi of nursery grapevines (Marais, 1980), our results suggest that a dramatic shift in the status of soilborne pathogens may have occurred over the last 20-30 years. A possible explanation for the low occurrence of Phytophthora in nurseries may be due to the extensive use of chemicals against downy mildew of grapevines, since some of the systemic chemicals applied to the foliage against downy mildew, especially the phosphonates, can be translocated downwards through the roots and thereby control Phytophthora root rot of grapevines (Marais & Hattingh, 1986). Furthermore, different methods of cultivation and propagation of nursery material could also contribute to the apparent change.
Pythium was more frequently obtained on 99 Richter than on 101-14 Mgt during this study. This was also the case previously (Marais, 1988).
The road ahead
Although the results of this study indicate that there appears to have been a shift in the status of soilborne pathogens in nurseries, only three sites in the Wellington production area were studied and a wider investigation is advisable to establish the relative distribution and significance of the pathogens. Variation in the incidences of Cylindrocarpon, Fusarium and Rhizoctonia, was observed between nurseries, where some nurseries had higher incidences of these fungi than other nurseries. Phytophthora occurred only in the third nursery, and Sclerotium only in the second nursery. These results support the case for a more comprehensive survey in the different production areas of grapevines in the Western Cape Province. Results of a larger survey should also elucidate critical areas of research and provide a basis for the development of disease management strategies for sustainable production of nursery vines.

FIG. 3. Incidence of fungal genera occurring in roots of diseased plants collected at three nurseries during the surveys.
Means within a fungal genus followed by the same letter do not differ significantly (P=0.05).
Incidence calculated as follows: Number of tissue pieces yielding the specific fungal genus / the total number of tissue pieces plated x 100.

FIG. 4. Incidence of fungal genera occurring in roots of diseased plants collected at the nurseries during three surveys.
Means within a fungal genus followed by the same letter do not differ significantly (P=0.05).
Incidence calculated as follows: Number of tissue pieces yielding the specific fungal genus / the total number of tissue pieces plated x 100.

FIG. 5. Incidence of fungal genera occurring in roots of diseased plants of the two rootstocks collected at the nurseries.
Means within a fungal genus followed by the same letter do not differ significantly (P=0.05).
Incidence calculated as follows: Number of tissue pieces yielding the specific fungal genus / the total number of tissue pieces plated x 100.

Acknowledgements
This research was funded by Stellenbosch University, Winetech (Project US/PP 02/2001), and the National Research Foundation. We thank Dr Paul Fourie and Mr Jan van Niekerk of the Department of Plant Pathology, University of Stellenbosch, for assistance during the project.
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