Abstract
Phylloxera has been an important pest in California since its discovery in 1880 within Sonoma and Napa counties. Phylloxera-resistant rootstocks and germplasm were selected from American grape species native to the eastern United States. Breeding programs to develop improved phylloxera-resistant rootstocks were started in the late nineteenth century. Resistance to phylloxera has been reported to be controlled by several genes. To focus on one aspect of resistance to phylloxera, a greenhouse screening method was used to observe absence/presence of root nodosities produced by phylloxera. Only one source of phylloxera biotype A from Fresno County was used to reduce the complication of various biotypes. Grape rootstocks with known field reaction to phylloxera were evaluated to test the reliability of the greenhouse test. A design II mating factorial cross was made between male and female rootstocks with a range of resistance to susceptibility. The reaction of their progeny to phylloxera was observed in the greenhouse. All populations segregated for resistance/susceptibility with a few exceptions. Dog Ridge crossed with two susceptible genotypes gave all susceptible offspring. Kober 5BB crossed with susceptible or resistant genotypes gave all resistant offspring. The segregation of resistance to nodosity development could be explained by two complementary dominant genes in most families.
Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major pest of grapes. It attacks both leaves and roots, but the most important damage occurs on the roots of Vitis vinifera wine, table, and raisin grapes. Native American grape species have been used to develop resistant rootstocks for the control of phylloxera. The initial work for breeding phylloxera-resistant rootstocks was conducted in Europe after the introduction of phylloxera during the late nineteenth century, and many of the rootstocks produced in Europe are now used in the United States. There is a need for the development of rootstocks with a wide range of adaptability not only because of phylloxera but also because of the variety of soil pests, diseases, and soil conditions.
The use of field trials in a breeding program to evaluate rootstocks for phylloxera resistance is time-consuming and impractical for evaluating seedling populations. Greenhouse tests have been used to rapidly evaluate numerous rootstocks and they adequately reflect their field reaction (Boubals 1966a). Plants were inoculated four times with massive infestations on a small number of roots and evaluated after approximately four months (Boubals 1991). The level or resistance or susceptibility was based on the highest grade of susceptibility found in all the replications. Greenhouse tests have also been used to observe phylloxera development and rootstock reaction to phylloxera (King and Rilling 1985). Phylloxera root damage has been classified as nodosities and tuberosities. Nodosities are the swellings on the young root tips and are the first sign of infection. Tuberosities are galled areas on lignified roots and are observed after longer periods of phylloxera infection. Tuberosities are critical indications of susceptibility in long-term field performance evaluations of known rootstocks. Some of the field trials in the late 1800s scored resistance on a 0 to 20 scale, with 20 being immune without the formation of nodosities or tuberosities (Viala and Ravaz 1903). Plants rated 16 to 19 had only a few nodosities and no tuberosities, indicating that the nodosities were the first indicator of susceptibility. More severely infected plants had both nodosities and tuberosities. Nodosities occurred when tuberosities were present, but the reverse was not always true and resistance was based on the relative number and size of nodosities (Husmann 1930). Rootstocks that resist nodosity feeding will also inhibit tuberosity formation, making the lack of nodosities the highest level of resistance (Forneck et al. 2001).
Resistance to phylloxera based on number of tuberosities was determined to be controlled by several genes (Boubals 1966b); the majority of seedlings produced in crosses between resistant rootstocks were very resistant. Vitis vinifera was considered homozygous susceptible and expressed partial dominance of resistance or susceptibility at varying degrees, depending on the resistant species it was crossed with (Boubals 1991). When Ganzin 1 (synonym, AXR 1) rootstock failed in Napa Valley, California, a new phylloxera strain biotype B was identified as able to reproduce more quickly on Ganzin 1 then biotype A (Granett et al. 1985). Laboratory tests of 118 phylloxera populations collected throughout California grapegrowing regions showed that only two biotypes were identified based on life cycles from Cabernet Sauvignon and Ganzin 1 root pieces (De Benedictis and Granett 1992). In tests of a number of rootstocks with biotype A, rootstocks rated as resistant or immune were almost always deemed resistant in European evaluations (Granett et al. 1987). In the development of new phylloxera-resistant rootstocks, it is important first to understand the inheritance of resistance to the most common phylloxera biotype and then to study additional biotypes. Biotype A was tested as it is most common in the San Joaquin Valley, the major table and raisin production area of California. A rapid screening method to choose individuals with the highest level of resistance is needed to screen high numbers of seedlings. Understanding the level of resistance transmitted by parents is important in planning crosses for the development of new rootstock hybrids. This study was undertaken to evaluate the greenhouse testing method and the propensity of nine rootstocks to transmit phylloxera resistance to their offspring.
Materials and Methods
Grape rootstocks with known levels of resistance or susceptibility were evaluated to determine the reliability of the greenhouse screening method. The tests were repeated three years on the parents used to create the seedling populations (Table 1⇓). Ganzin 1 was inoculated to determine the biotype of phylloxera used in the tests. Other rootstocks with known resistance were also tested. The phylloxera used in these tests did not survive or produce nodosities on Ganzin 1 (Table 1⇓), and therefore were determined to be biotype A. Only biotype A was used to lay the groundwork for a basic understanding of inheritance of resistance to phylloxera. The 3- to 4-node dormant cuttings were rooted, and when roots were 5 to 10 cm long the cuttings were planted in 15-cm diam pots (2,296 cm3). The planting medium consisted of 1 part Sunshine Mix #4 (Sun Gro Horticulture, Vancouver, Canada) to 1 part coarse perlite. Phylloxera were collected from commercial Ruby Seedless vineyards on their own roots east of Reedley, Fresno County, California. The cuttings were inoculated at the time of planting by placing three or four root pieces, 3 to 4 cm long, containing nodosities with high numbers of adults, larvae, and eggs next to the roots. The goal was to inoculate with high numbers of phylloxera on a low number of roots to establish phylloxera on susceptible rootstocks. Temperatures fluctuated between 16 and 27°C in the greenhouse. Plants were hand watered as needed for healthy root growth and subsequent phylloxera development. The roots were evaluated for nodosities, phylloxera, and eggs. Severity of infection was rated on a 1 to 4 scale (1 = no nodosities, 2 = 1 to 50 nodosities, 3 = 50 to 100 nodosities, 4 = >100 nodosities) so comparisons could be made with results in the literature. The score of the most heavily infected replication was used as the reaction of the rootstocks to phylloxera. Average scores are also reported to define moderately from highly susceptible rootstocks and determine if they transmit resistance or susceptibility at different rates. The rootstocks were evaluated 14 weeks after inoculation the first year (1996), after 12 months with three different inoculations the second year (1997), and 6.5 months after inoculation the third year (1998).
To create the design II mating factorial cross, five female flowered rootstock cultivars were hybridized with four male flowered rootstocks in all combinations. The rootstock cultivars ranged from resistance to susceptible. The nine rootstocks used with their parentage and field resistance are listed (Table 1⇑).
A maximum of 20 seedlings from each family in the design II mating factorial were evaluated as individual plants in 15-cm diam pots throughout the year after inoculation. To conserve space in the following years, plants were grown in 5.7-cm square pots (260 cm3). Leafy cuttings were made from field-grown plants in June and July by rooting them under intermittent mist in 100 Flexiplug (GrowTech, Roselle, IL). Four rooted leafy cuttings of each seedling were planted using the same soil mix as above. Before planting in the pots, a hole was made in the soil for the rooted cutting. Three or four Ruby Seedless root pieces ~1 cm long containing nodosities with phylloxera adults, larvae, and eggs were placed in the hole, after which the cutting in the rooting sponge was planted. The plants were evaluated 6 to 8 weeks after inoculation. The plants were reinoculated and evaluated again after 6 to 8 weeks. When possible, a total of 80 plants per family were evaluated in the greenhouse. Thompson Seedless and 10-23B (a selection of V. doaniana that has very good nematode resistance but is susceptible to phylloxera) were included in all tests as susceptible controls. Watering was very critical in these small pots, so they were hand watered as needed to encourage actively growing roots. These conditions are necessary for proper phylloxera feeding and development of nodosities and egg-laying adults to occur for good evaluation of the resistant/susceptible reaction. The same rating scale as above was used. However, any individual that had a rating of 2 or greater (i.e., >3 to 5 phylloxera surviving with the production of nodosities on any replicate) was considered susceptible. Only nodosities were evaluated, as they are the first form of infection to occur and produce the most rapid results. Nodosities are also visible over a longer period of time than eggs, larva, or adults, irrespective of the insect’s life cycle. Seedlings that were rated as 1 were evaluated again the second year to make sure they had not escaped infection.
Results and Discussion
The resistant/susceptible reactions for the rootstock parents used in the design II mating factorial are shown (Table 2⇓). Additional rootstock cultivars were tested and are also reported. The rootstocks 101–14 Mgt, Teleki 5C, Paulsen 1103, and Kober 5BB reported in the literature as resistant showed no nodosities or phylloxera survival in the greenhouse tests. Ramsey (synonym, Salt Creek) was resistant all three years in the greenhouse, which agrees with reports for biotype A in root bioassay (Granett et al. 1987) and in France (Boubals 1991) and Australia (Hardie and Cirami 1988). In a dual-tissue culture test, Ramsey had high levels of initial nodosity development but low reproduction and few survivors and was rated as resistant (Kellow et al. 2002). The rootstocks reported in the literature as susceptible or with low resistance showed development of nodosities and phylloxera reproduction. Thompson Seedless and Dog Ridge had the highest phylloxera ratings every year. Dog Ridge was resistant in root bioassays with biotype B (Granett et al. 1987), resistant but not immune in field trials (Pongracz 1983), and susceptible in France (Boubals 1966a) when rated for tuberosities. St. George also supported phylloxera populations in all tests, although not as high as Thompson Seedless or Dog Ridge. St. George was reported to have low resistance in the field and noted for its ability to support phylloxera, but no report of failure in the field was noted (Wolpert et al. 1994). However, St. George was resistant in root bioassays with biotype A and B (Granett et al. 1987) and resistant but not immune when evaluated based on tuberosities (Pongracz 1983, Boubals 1966a). Couderc 1613 was inconsistent in our tests, with phylloxera developing in one of the three years tested. This finding agrees with the varying levels of resistance that have been reported for Couderc 1613 depending on the evaluation method and phylloxera source/biotype. Couderc 1613 was resistant to biotype A and B in root bioassays (Granett et al. 1987), moderately resistant in field trials (Kasimatis and Lider 1975), with low resistance in greenhouse tests where nodosities but not tuberosities formed (King et al. 1982), or susceptible in greenhouse and field tests (Boubals 1966a, 1991, Wolpert et al. 1994).
The results for all families of the design II mating factorial are shown (Table 3⇓). Susceptible parents produced high levels of susceptible offspring except when they were hybridized with Kober 5BB, which produced almost 100% resistant progeny regardless of the resistant or susceptible parent with which it was hybridized. It is interesting that Kober 5BB gave this many resistant progeny since it was reported to only be tolerant in Europe, where phylloxera reproduced on its roots (Börner 1942). The female parents Ramsey, Couderc 1613, and 101–14 Mgt were intermediate in their production of resistant seedlings. Ramsey produced a higher percentage of resistant progeny than Couderc 1613 or 101–14 Mgt, especially in crosses with the susceptible parents Thompson Seedless and St. George. Dog Ridge gave no resistance. The male parent Teleki 5C transmitted a high percentage of resistance, although not as high as Kober 5BB. Teleki 5C gave a higher percentage of resistance than Ramsey when crossed to susceptible parents, but was the same when crossed with resistant parents. The family size of Paulsen 1103 was generally too small to draw conclusions; however, it had levels of resistance similar to Teleki 5C. Thompson Seedless and St. George gave no resistance when crossed to Dog Ridge. St. George had more propensity to increase the percent resistant progeny compared to Thompson Seedless when they were hybridized with resistant rootstocks.
Based on observations here, resistance to phylloxera-induced nodosity development appears to be controlled by two complementary dominant genes. Kober 5BB is homozygous for both genes as all its progeny were resistant, even when crossed with the susceptible parents Dog Ridge and Thompson Seedless. When susceptible cultivars were intermated (Dog Ridge x Thompson Seedless or St. George), they produced almost 100% susceptible progeny, as expected. The best fit of the observed ratios for resistance compared to expected ratios based on a two complementary dominant gene model was determined by X2 (Table 3⇑). The observed ratios fit the expected ratios expected in three cases. Boubals (1966b) reported that resistance was controlled by several genes when hybridizing various rootstocks of similar species, which is consistent with findings here.
Other grapes species have shown various inheritance patterns for resistance. An average of 51.6% (range 39.7 to 65.4%) phylloxera-resistant seedlings occurred in V. vinifera x Muscadinia rotundifolia (VR) F1 families (Bouquet 1983). Resistance was classified as no formation of tuberosities on roots or nodosities on rootlets. The occurrence of resistant plants in the BC1 progenies was less than 20%. A genetic scheme consisting of a semidominant gene that is homozygous in M. rotundifolia and controlled by three modifier genes in the F1 VR families was suggested. The low level of resistant plants in the BC1 could be explained if the resistant gene is located on the Muscadinia chromosome, which has a low chance of paring with a vinifera chromosome. Others have reported that VR hybrids were resistant and suggest M. rotundifolia carries a dominant gene for resistance (Firoozabady and Olmo 1982). The seedling populations segregated for resistance but percentage resistance was not reported.
Vitis cinerea Arnold, a selection not included in this study, was found to have complete resistance to phylloxera and was hybridized with V. riparia 183G to create the resistant rootstock Börner (Becker 1988). Rootstocks hybridized with Börner ranged widely in percent resistant progeny. Resistance to both root and leaf gall forms ranged from the highest of V. berlandieri Resseguier #1 (75% resistant), Kober 125 AA (48%), Dog Ridge (35%), Binova (28%), to less than 5% resistant progeny from Kober 5BB and 101–14 Mgt (Schmid et al. 2003). This study concluded that resistance originates from more than one gene and the parent hybridized with Börner influenced the proportion of resistant hybrids. The inheritance of resistance appears to be different from results here. The lower levels of resistance from Kober 5BB may be due to the occurrence of biotypes of phylloxera that overcame its resistance. A single quantitative trait locus on linkage group 13 has been found to associate with very low nodosity numbers (resistance) originating from V. cinerea in Börner rootstock hybridized with V. vinifera (Zhang et al. 2009). Two makers tightly linked with resistance as scored by counting nodosities after inoculation were identified. The resistant ratios of the progeny varied in the two years observed because not all were tested each year. In 2006 and 2007 the resistant to susceptible ratios were 31:96 and 71:161, respectively. These ratios are similar to the 1:3 ratio obtained from Couderc 1613, 101–14 Mgt crossed to Thompson Seedless, but less than the 3:5 ratio obtained for Ramsey crossed to Thompson Seedless. The most reliable indicator of the quantitative resistant phenotype was the maximum number of nodosities per individual plant.
Resistance to the formation of nodosities in an F2 progeny of V. vinifera Aramon x V. rupestris Ganzin 1 (a remake of AXR 1) segregated in a 1:7 ratio (Roush et al. 2007). Resistance was rated as less than 6.2 nodosities per plant, the level found on the resistant parent, suggesting that at least two loci are involved. This level of resistance does not seem to be as high as that found for the rootstocks tested in this study or from Börner rootstock (Zhang et al. 2009). It was unclear whether one or two genes control tuberosity formation, which was studied separately from nodosity formation. In both cases, resistance was recessive and separate mechanisms for the control of nodosity or tuberosity formation were suggested.
There was good correlation between the 15-cm and 5.7-cm pot tests except for four families the first year. When these families were evaluated the second year in 5.7-cm pots, the correlation was similar to the 15-cm pots but with slightly more resistant observations. This inconsistency may be due to the low number of seedlings tested in 15-cm pots or susceptible individuals escaping because of the increased difficulty in controlling growing conditions in 5.7-cm pots. There are advantages and disadvantages of testing in 15-cm and 5.7-cm pots. The 15-cm pots can be accurately evaluated from seedlings in 1 to 2 years. However, pot size greatly limits the number of plants that can be screened and occupies the greenhouse with the same plants for one to two years. The 5.7-cm pots can be evaluated in 3 to 6 weeks and thus two to three tests can be conducted in one year. Since the 5.7-cm pots tend to be more sensitive to environment factors, the number of replicates per plant probably should be increased to eight for more accurate results.
Conclusions
The greenhouse test can be used to differentiate resistant from susceptible individuals as seen in the literature. Rootstocks susceptible to phylloxera did not transmit resistance to their seedlings while those with low resistance transmitted a low percent of resistance to their offspring. Resistant rootstocks varied in the percent resistance transmitted to their offspring and Kober 5BB was the best, producing 99 to 100% resistant offspring even when crossed with susceptible rootstocks. In most families, resistance to nodosity development could be explained by two complementary dominant genes.
Footnotes
Acknowledgments: This project was financed in part by a grant from the Viticulture Consortium West.
The author thanks Richard L. Emershad and Erica Crouch for past and ongoing technical support in the design of the greenhouse, plant care, and phylloxera evaluation.
- Received September 2009.
- Revision received January 2010.
- Accepted February 2010.
- Published online June 2010
- Copyright © 2010 by the American Society for Enology and Viticulture