RAPD Assessment of California Phylloxera Diversity. Molecular Ecology 4:459464.)

California grape phylloxera more variable than expected

Jeffrey Granett 0 Andrew Walker P John De Benedictis GenineFong o Hong Lin P Ed Weber

Many strains of grape phylloxera phylloxera’s biology, its life cycle and now have been identified in Cali- how grape species and rootstocks re- fornia vineyards. This variability sist its feeding. may be the result of multiple intro- This paper reviews our recent dis- ductions of this pest or of evolu- coveries that the DNA and feeding f tion of new strains on susceptible behavior of different collections of -G* or weakly resistant rootstocks. California phylloxera are relatively di- 8 1 verse. The discovery of diversity in 0 Thus own-rooted vines, weakly re- -J sistant rootstocks and those with biological populations is not novel, but the amount we found in California Grape phylloxera on rootstock. The minute V. vinifera parentage should not aphidlike insects feed on the root, causing a was unexpected. Because phylloxera be used in phylloxerated areas. In gall to form. The expanding and cracking gall were supposedly introduced into Cali- limits the root’s ability to take up water and addition, because of the observed fornia a relatively short time ago, and nutrients and allows rot organisms to invade. variability, quarantines are inef- they only reproduce asexually on the fective in preventing the occur- roots, we expected a relatively low are rarely damaged by its feeding. rence of biotype B phylloxera, as level of diversity. Other Vitis species, from parts of the it appears to evolve independently The variability in phylloxera’s abil- world where phylloxera are not na- in different areas. ity to feed on different grape species tive, did not evolve this resistance and and cultivars is of central importance are highly susceptible to damage. Vitis Grape phylloxera, Daktulosphaira to the use of rootstocks. Vitis species vinifera, the European grape species vitifoliae (Fitch), is one of the worlds and the rootstocks bred from them are from which most of our cultivated most important viticultural pests. This not uniformly resistant to grape phyl- grapes are derived, is one such suscep- minute aphidlike insect lives on grape loxera, nor are grape phylloxera types tible species. roots and foliage, and is most damag- uniformly aggressive on a given spe- Phylloxera were unintentionally ing to grape species that are suscep- cies or rootstock. As is common imported to Europe in the mid-1800s. tible to its root feeding. This feeding throughout biology, there is variability Vineyards before and during this pe- causes a swelling of the root that de- in both the plant and the insect. Many riod were planted on their own roots, velops into a gall and nourishes the questions have emerged: What gener- and phylloxera soon decimated them. insect. As the galling develops it ex- ated the diversity? Are other root- Numerous insecticides and other con- pands and cracks, wounding the root stocks in imminent danger from a trol measures were attempted, but and potentially allowing the entry of damaging biotype? How does this di- none proved effective at limiting rot organisms. The combination of versity affect the breeding and selec- phylloxera’s rapid spread. By the phylloxera’s limiting effect on the root tion of new rootstocks? 1870s, French viticulturists observed system’s ability to take up water and To begin addressing these ques- that the American grape species nutrients and the effect of rot organ- tions, we describe experiments to were not susceptible to phylloxera. isms eventually kills infested vines. evaluate the variability of grape phyl- Planchon and Laliman developed the Phylloxera can devastate a susceptible loxera in California and elsewhere. We idea that these root systems could be vineyard in just a few years. also review what is known about phyl- grafted with V. vinifera scion cultivars Grape phylloxera have been de- loxera aggressiveness from other to produce a perfect plant - one stroying vineyards around the world viticultural regions. whose root system was resistant to for the past 140 years. Fortunately, phylloxera, but whose top produced phylloxera-resistant rootstocks have Phylloxera native to the Americas commercially desirable grapes. Breed- been available and have effectively de- Grape phylloxera are native to the ers were soon crossing American Vitis fended vineyards against this pest for Americas, where Vitis species serve as species to produce rootstocks with about 110 years. Despite this long his- hosts. These American Vitis species phylloxera resistance, good horticul- tory, many questions remain about have coevolved with phylloxera and tural characters (such as rooting and

CALIFORNIA AGRICULTURE, JULY-AUGUST 1996 9 grafting ability) and adaptation to Eu- ropean soils and climates. Hundreds of rootstocks were produced, tested and deployed throughout Europe. As phylloxera was spread to the rest of the viticultural world, these rootstocks followed in the insect’s devastating wake and are still effectively used today. AxR#1 was one such rootstock developed in the late 1800s. It is a hybrid between the V. vinifera cultivar Aramon and the resistant grape species V. rupestris. This combination was made to produce a rootstock with ex- cellent drought and lime tolerance, the easy propagation of V. vinifera and the phylloxera resistance of V. rupestris. AxR#l was used in Europe and South Africa after its release, but its V. vinifera parentage caused it to succumb to phylloxera after 10 to 30 years of use. Phylloxera was first found in Cali- fornia in the 1860s, and viticulturists here used the European rootstocks to combat the pest. Rootstock trials were initiated in 1905 to determine which rootstocks were best suited to California’s grape-growing regions. By Vines on the left are grafted to the rootstock llOR, which is resistant to phylloxera 1958 the collected data were used to biotype B. Vines on the right are grafted to AxR#l. recommend AxR#l as well adapted to most of California’s vineyard soils, cli- mate, water conditions and scions. replanting this acreage using root- tional designations based on the be- Based on this recommendation, AxR#l stocks more resistant to phylloxera havior of phylloxera, not necessarily was used in 60 to 70% of the Napa and than AxR#l. on genetic differences. Sonoma County plantings that oc- In addition to biotypes A and B, curred in the 1960-1980 planting Phylloxera biotypes and diversity other types of phylloxera have been boom. Biotype B phylloxera were studied found on rootstocks other than AxR#1. However, AxR#1 began to collapse by comparing their survival and re- Some of these phylloxera developed under phylloxera pressure in 1983, production to those of the less aggres- relatively high populations, but did and by 1989 it was clear that AxR#l sive biotype A in laboratory-based as- not appear to affect the vines. These was not adequately resistant to Cali- says (bioassays) using root pieces of phylloxera behave differently than fornia phylloxera (see California Agri- AxR#l and Cabernet Sauvignon (see biotypes A and B in the root bioassays. culture March-April 1991). The decline California Agriculture Jan-Feb 1987). of AxR#1 was attributed to a new type The two biotypes were defined by of phylloxera, designated biotype B to their performance in these bioassays: distinguish it from phylloxera inca- biotype A phylloxera are able to grow pable of killing AxR#l, termed bio- and reproduce well on V. vinifera type A. roots, but do not perform well on Biotype B has been found through- AxR#l. In contrast, biotype B phyllox- out Napa and Sonoma counties and in era are able to grow and reproduce Lake, Mendocino, San Joaquin, Sacra- well on both root types. In all cases, mento, Santa Clara and Alameda phylloxera evaluated from declining counties. By the end of 1995, over AxR#l vineyards have been biotype B 12,000 acres of vineyards in Napa and Neither biotype A nor B is able to Sonoma counties had already been re- grow well on rootstocks such as Har- Fig. 1. Yearly total of AxR#l vineyard acre- moved as a direct result of biotype B mony, 5C, 3309, St. George, 110R and age removed due to biotype B phylloxera phylloxera (fig. 1).Grape growers are many others. Thus biotypes are func- infestation, Napa and Sonoma counties.

10 CALIFORNIA AGRICULTURE, VOLUME 50, NUMBER 4 feeding on mature roots is most damaging in the field. DNA was extracted from 13 of our lab-based phylloxera colonies repre- senting biotypes A and B and several strains. Extractions were made from 200 to 300 phylloxera eggs per colony, which were surface sterilized and sub- jected to PCR-RAPD reactions (poly- merase chain reaction-randomly am- plified polymorphic DNA). RAPDs depend on the ability of randomly generated 10-base-pair DNA sequences (primers) to match with the corresponding sequences in the phylloxera DNA. The lengths of DNA between attachment locations for the primers are spliced out and replicated many times. RAPD results are viewed on agarose gels as bands of different- sized DNA, the smaller bands moving further in the gel (fig. 2). Bands are quantified by their position and their presence or absence at each possible location. When analyzed, the results estimate how closely related (or unre- lated) the phylloxera DNAs are to each other (Fong et al. 1995). Most of the vines in this vineyard are grafted to AxR#l. The healthy vine in the center is RAPD analyses using 10 different grafted onto St. George, a rootstock that is resistant to biotype B. primers were run three times on the 13 DNA samples to ensure reaction re- producibility. Clear RAPD bands from Because they were not killing their Phylloxera development was exam- the three replicates were then scored host rootstocks, but instead appeared ined in petri dishes by placing single as either present or absent and com- to have adapted to them, the new eggs onto cleaned root pieces (about pared. There was no amplification of phylloxera types were designated as .25 inch by 2 inches) of Cabernet negative controls consisting of all "strains" in order to separate them Sauvignon, or onto roots of the root- RAPD ingredients minus phylloxera from destructive biotypes. stock the phylloxera were collected DNA. We have recently examined the from. Insect development and repro- DNA of these various phylloxera duction was monitored under con- Genetic diversity within biotypes types and determined that there is trolled conditions. Comparisons of col- Table 1 shows some of the biotypes considerable diversity within and lected colonies were based on and strains we have found to date. The among the designations used to repre- differences in the percentage survival term "strain" has been used to desig- sent feeding behavior. of phylloxera to the adult stage; the nate colonies that do not respond in time required for individuals to reach the same manner as biotypes A and 8. DNA analysis of phylloxera the adult stage; and, once adults, their We are now using this term more pre- Phylloxera colonies were collected rate of egg laying. This information cisely to designate genetically uniform from throughout California. Initially was combined to produce an egg mul- colonies derived from a single egg; we collected phylloxera only from tiplication index (EMI). This index thus there can be several strains of bio- vineyards planted on AxR#l or their gives a measure of how many phyllox- type A or B based on geographic ori- own roots. Bioassay results from these era will be present in the next genera- gin, differences in bioassay data or collections suggested that we were tion, and therefore estimates a given RAPD data. missing a large proportion of the colony's ability to damage a host. EM1 Our discovery of multiple strains of insect's variability, so we expanded values have been calculated for many genetically different biotype B phyl- our collections to rootstocks other than phylloxera colonies on a variety of loxera suggests that biotype B has AxR#l. Phylloxera from other states rootstocks. Phylloxera survival on cal- been selected forjn AxR#1 vineyards and countries, collected by various col- lus or newly formed roots was not en- more than once. There is sufficient leagues, were also examined. tered into the EM1 formula, because variability, due either to multiple im-

CALIFORNIA AGRICULTURE, JULY-AUGUST 1996 11 ing was sup- on roots of Cabernet Sauvignon, and porting the did not establish on AxR#1; it was able ph ylloxera. to survive on the roots and callus of These three 5C and S04. How well these German strains devel- phylloxera are adapting to V. berlandieri oped on both X V.riparia rootstocks is under investi- Cabernet gation. Sauvignon We first used RAPD analysis in an and AxR#l in effort to "fingerprint" biotypes. How- root bioassays ever, we found that strains within bio- and had types were genetically different. For higher EM1 example, RAPD patterns of the bio- values than type A colonies listed in table 1 ranged biotype A on from very similar to very distinct. This AxR#1. They was expected, but RAPD results of the are also dis- biotype B colonies in table 1, and other Fig. 2. Agarose gel electrophoresis of tinguished by B types we have tested, indicate that RAPD products from paired comparisons of biotype A vs. biotype B from Rutherford their ability to maintain themselves on they are similarly diverse. The level of using 9 Operon primers. The lane on the several non-vinifera rootstocks by feed- genetic diversity found among phyl- far left is a 123 bp DNA molecular ladder; ing on callus and immature roots. This loxera colonies was relatively high, Lanes 1 and 2 = OPG3, Lanes 3 and 4 = OPG4, Lanes 5 and 6 = OPGl 0, Lanes 7 sort of feeding is not indicated in EM1 considering that Californian phyllox- and 8 = OPA4, Lanes 9 and 10 = OPA9, values, which are based on growth era are supposed to have been intro- Lanes 11 and 12 = OPAl 0, Lanes 13 and and reproduction on lignified l-year- duced 130 years ago and that they are 14 = OPAl1, Lanes 15 and 16 = OPA12 and old roots. Strain 4 was found on se- asexual. This level of diversity was not Lanes 17 and 18 = OPBl. Differences between the two biotypes are indicated with verely declining Harmony vines in an expected, and has prompted investiga- arrows. Oakville rootstock trial (18 vines in tions into the roles that multiple intro- one replicate of the experiment). Be- ductions, sexual recombination and portations or to rapid evolution, to cause of strain 4's ability to damage mutation may have on phylloxera suggest that phylloxera will continue Harmony, it has been designated as a variability. to adapt to weakly resistant root- biotype. stocks. Thus a phylloxera quarantine An apparently native strain of phyl- American Vitis resistance designed to prevent the spread of ag- loxera has been found on V. girdiana in The origin of grape phylloxera vari- gressive strains into an AxR#1, or Death Valley. These phylloxera have ability is unknown, but there are pos- other V.vinifera-containing rootstock only been found on leaves, and we sible explanations. The variability vineyard, will not prevent damage for have not been able to establish them could reflect the initial mix of popula- long. Local quarantines in nonphyllox- on any roots. Thus they are quite dif- tions brought to California as hitchhik- erated areas of the state may still be ferent from the other phylloxera ing insects on infested Concord grapes useful because natural spread is slow. strains we have tested. We have not and rootstocks brought with settlers However, because natural and inad- yet compared Death Valley phylloxera from the eastern United States, and on vertent spread is possible, planting of DNA with that from other strains. V. vinifera vines from Europe. It is also phylloxera-susceptible rootstocks in Phylloxera tested from other states, possible that an unobserved sexual soils capable of supporting phylloxera including Florida, Kansas, Missouri, phase of the Californian phylloxera's is not recommended. New York and Oregon, have been life cycle may be generating variability Strain 1 was isolated from Free- type A, but their RAPD patterns are and allowing the insects to rapidly dom growing in an Oakville root- very different from those of Califor- adapt to new hosts. Some aphids are stock trial. The vines were not de- nian phylloxera. able to rapidly adapt to new hosts or clining, but this strain has survived We also tested German phylloxera to develop resistance to pesticides by and reproduced on young roots and collected from declining vineyards altering their genetic makeup during callus of 5C, St. George, Dog Ridge, planted on the rootstocks 5C, 5BB and asexual reproduction. This may also 101-14Mgt, Harmony and Freedom 125AA (all V. berlandieri X V. riparia occur with the phylloxera root aphid. in laboratory bioassays. Strain 2 was rootstocks). Although new, more ag- The variability we are observing could isolated from St. George in Healdsburg gressive phylloxera have been associ- also reflect relatively high mutation and has survived and reproduced on ated with decline, the soils are shallow rates, although such rates have not callus and young roots of 5C and 101- and rocky and the vines have been wa- been observed in related insects. 14Mgt. Once again, the vines from ter stressed. As expected, RAPD tests The resulting variability allows new which Strain 2 was isolated were not found this strain to be very different phylloxera strains to evolve as they declining. Strain 3 was isolated from from all tested Californian strains, and adapt to various rootstocks. In Califor- Dog Ridge in Lodi. The vines were de- it performed differently in bioassay nia vineyards we have observed declining, but it is likely that scion root- tests. This strain was slow to establish cline due to phylloxera only on

12 CALIFORNIA AGRICULTURE, VOLUME 50, NUMBER 4 rootstocks with V.vinifera parentage, half vinifera and has failed to resist Harmony and Freedom have simi- but we have observed strains that ap- phylloxera in many countries. We lar parentages, which include a small pear to be better adapted to rootstocks have also seen failure in Harmony, a percentage of vinifera. Harmony has with pure American Vitis species par- rootstock with a small percentage of been susceptible in root bioassays, and entage. It is possible that these newly vinifera parentage. It is not understood we have observed decline due to phyl- adaptive strains could become damag- how the American Vitis species resist loxera in the field, yet Freedom exhib- ing biotypes. phylloxera, but rootstocks produced its resistance in root bioassays, and no Phylloxera biotypes have evolved with only American Vitis species have cases of vineyard decline have been re- in California, and anything that in- been effective against phylloxera for ported. In each of these cases, one of creases the genetic variability in a over 100 years. Thus it is prudent to the paired rootstocks has declined in population increases the potential for recommend that only rootstocks with California while the other has not. selection of new biotypes. Therefore, American Vitis species parentage be de- Given that we know little about phyl- keeping field populations low de- pended upon for phylloxera resistance. loxera resistance, and that phylloxera creases the potential for selecting bio- There are anomalies to this recom- are variable and adapting to new types. Phylloxera populations can be mendation. Like AxR#1, 1202C is half hosts, 1202C, 039-16 and Freedom reduced by fumigating vineyard vinifera, yet it has performed well in cannot be recommended solely on the ground prior to replanting with root bioassays and no confirmed cases basis of their phylloxera resistance. grapes; removing the majority of of field decline have been reported in Finally, our data on strains 1, 2,3 grape roots before replanting; leaving California (although it has declined in and 4, and the German discoveries the site fallow from at least fall to late Europe). 039-16 and 043-43 have very suggesting phylloxera damage in non- spring; and replanting using root- similar parentages (V.vinifera X vinifera rootstocks, indicate that phyl- stocks with the highest levels of phyl- Muscadinia rotundifolia),yet 039-16 has loxera susceptibility may exist in pure loxera resistance. exhibited very high resistance in root American species rootstocks. If so, the A survey of the world rootstock lit- bioassays, while 043-43 appears sus- stability of these rootstocks may not be erature suggests that only rootstocks ceptible. We have found 043-43 de- eternal, and we should be prepared for that have partial vinifera parentage are clining due to phylloxera, but there are phylloxera strains that are better susceptible to phylloxera. AxR#l is no reports of 039-16 declining. adapted and potentially damaging in the future. The way to combat this threat is through knowledge of phylloxera biology and its interactions with Vitis. Then we can predict how potentially damaging phylloxera may become and better guide the breeding of new resistant rootstocks.

J. Granett and J. De Benedictis are Ento- mologists, Department of Entomology, UC Davis; A. Walker is BreederlGeneti- cist and Viticulturist, G. Fong is Gradu- ate Student, and H. Lin is Postgraduate Researcher, Department of Viticulture and Enology, UC Davis; and E. Weber is Viticulture Farm Advisor, UC Coopera- tive Extension, Napa County. We wish to thank the American Vine- yard Foundation, the California Table Grape Commission, The Wine Group, lnc. and the California Grapevine Rootstock Improvement Commissionfor funding this project. Wealso gratefully acknowledge fundingfrom a specific cooperative agree- ment with the USDAIARS-Fresnofor phylloxera research.

For further reading: Fong, G., Walker, M.A. and Granett, J. 1995. RAPD assessment of California phylloxera diversity. Molecular Ecology 4:459464.)

CALIFORNIA AGRICULTURE, JULY-AUGUST 1996 13