Armillaria root disease affects orchards in all Juglans regia (Persian walnut)-growing regions of California (Gardner and Raabe, 1963). The causal agent is Armillaria mellea (Basidiomycota, Physalacriaceae), which attacks walnut and other woody perennial horticultural crops (e.g., stonefruits) (Baumgartner et al., 2011). The pathogen kills and then decays woody roots, which in turn reduces crop yield and growth (Baumgartner, 2004), inhibits nutrient and water uptake (Baumgartner and Warnock, 2006), and eventually kills infected trees. The primary inoculum consists of mycelium within dead, woody roots left in the soil after infected trees are cleared. This inoculum is persistent in field soil; in orchards and vineyards, viable mycelium has been recovered from 10-year-old or older residual roots (Baumgartner and Rizzo, 2002; Rizzo et al., 1998). Attempts to eradicate inoculum by soil fumigation indicate that penetration by methyl bromide or carbon disulfide is efficacious within the top 0.3 to 0.6 m of soil, but inoculum buried at greater depths is likely to persist (Bliss, 1951; Munnecke et al., 1981). The persistent inoculum and lack of effective preventive or curative methods limit yields throughout the life of an infected walnut orchard.
Walnut is not typically grown on its own roots in orchards in California. This is attributable in part to the susceptibility of J. regia to several widespread root diseases (e.g., Armillaria root disease, Phytophthora crown and root rot) (Mircetich et al., 1998). As such, 80% of walnut orchards are planted on Paradox rootstock, a seed-propagated hybrid of J. hindsii (Northern California black walnut) and J. regia, which is more vigorous, more resistant to some Phytophthora species, and more tolerant of soil salinity than J. regia (McGranahan and Catlin, 1987). Despite its widespread use, the resistance of Paradox rootstock to Armillaria root disease, crown gall, and Phytophthora crown and root rot is insufficient as evidenced by serious losses to the walnut industry from these root diseases (McGranahan and Leslie, 1990). Breeding efforts have focused on identifying sources of resistance among J. hindsii and other wild species of black walnut and crossing them with J. regia to develop new clonally propagated, disease-resistant Paradox rootstocks (Hackett et al., 2010). For example, RX1 (J. microcarpa × J. regia) is resistant to Phytophthora crown and root rot (Browne et al., 2010). A transgenic approach has also been used (Hackett et al., 2010), specifically to engineer rootstocks for RNAi-mediated resistance to crown gall (e.g., RR4 11A). However, there are no reports of identified resistance to A. mellea from controlled inoculations of J. regia, J. hindsii, or Paradox rootstocks in the greenhouse or field.
Field trials for Armillaria species require controlled inoculations after plants are established and then ≈10 years for sufficient evaluation (e.g., Guillaumin et al., 1989). To identify the most promising sources of resistance before establishing such trials, it is therefore critical to have a rapid and reliable screening assay. A slow and unreliable greenhouse assay has been a barrier to identification of Armillaria-resistant rootstocks for many fruit and nut crops despite repeated attempts at improvement by numerous scientists over the course of many decades (Baumgartner and Rizzo, 2006; Raabe, 1979; Thomas et al., 1948). A. mellea grows so slowly that the initial stages of the assay (inoculum preparation, establishment of infection) take one year (Redfern and Filip, 1991). The inoculum, which consists of wood fragments colonized by the pathogen, contains variable concentrations of mycelium and is surprisingly short-lived in the greenhouse, sometimes dying before an infection is established. This results in unequal challenge among replicate plants, which in turn confounds detection of differences between rootstocks. Furthermore, the greenhouse assay does not bring about repeatable symptoms or mortality (Singh, 1980). Without such measures of resistance, therefore, there are no quantifiable means of comparing rootstocks.
As an alternative to the unreliable greenhouse assay, we used a growth-chamber assay originally developed for grape (Baumgartner et al., 2010) for walnut plants micropropagated in a culture medium that supported both host and pathogen. Our objective was to evaluate six clonally propagated Paradox rootstocks (AX1, Px1, RX1, RR4 11A, VX211, Vlach) for resistance to Armillaria root disease. Paradox rootstocks were compared with a J. hindsii selection, W17, and a J. regia scion cultivar, Chandler, which are presumed to be Armillaria-resistant and Armillaria-susceptible, respectively, based on one field trial in which seedlings of both species, albeit different genotypes than W17 and ‘Chandler’, were planted in an orchard with a history of Armillaria root disease (Hendricks et al., 1978). In addition, the Juglans relative Pterocarya stenoptera (Chinese wingnut) was included for comparison, which is highly resistant to P. cinnamomi and P. citricola, and is thus used as a resistant control in screening for Phytophthora resistance (Browne et al., 2011).
Baumgartner, K., Bhat, R. & Fujiyoshi, P. 2010 A rapid infection assay for Armillaria and real-time PCR quantitation of the fungal biomass in planta Fungal Biol. 114 107 119
Baumgartner, K. & Warnock, A.E. 2006 A soil inoculant inhibits Armillaria mellea in vitro and improves productivity of grapevines with root disease Plant Dis. 90 439 444
Box, G.E.P., Hunter, W.G. & Hunter, J.S. 1978 Statistics for experimenters: An introduction to design, data analysis, and model building. Wiley, New York, NY
Browne, G.T., Grant, J.A., Schmidt, L.S., Leslie, C.A. & McGranahan, G.H. 2011 Resistance to Phytophthora and graft compatibility with Persian walnut among selections of Chinese wingnut HortScience 46 371 376
Browne, G.T., Schmidt, L.S., Bhat, R., Leslie, C.A., Hackett, W., Beede, B. & Hasey, J. 2010 Etiology and management of crown and root rots of walnut, Walnut Research Reports. California Walnut Board. p. 225–236
Buzo, T., McKenna, J., Kaku, S., Anwar, S.A. & McKenry, M.V. 2009 VX211, a vigorous new walnut hybrid clone with nematode tolerance and a useful resistance mechanism J. Nematol. 41 211 216
Guillaumin, J.J., Pierson, J. & Grassely, C. 1989 The susceptibility of different Prunus species used as stone fruit rootstocks to Armillaria mellea (sensu stricto). Seventh International Conference on Root and Butt Rots, Vernon and Victoria, British Columbia, Canada. p. 197–207
Hackett, W., Leslie, C., Grant, J., Lampinen, B., McGranahan, G., Anderson, K., Bob, B., Buchner, R., Caprile, J., DeBuse, C., Hasey, J., Manterola, N., Robinson, R., Kluepfel, D., Browne, G. & McKenry, M. 2010 Clonal propagation of walnut rootstock genotypes for genetic improvement, Walnut Research Reports. California Walnut Board. p. 65–83
Hendricks, L., LaVine, P., Mircetich, J. & Ramos, D. 1978 Differential susceptibility of walnut rootstocks to Armillaria root rot, Walnut Research Reports. California Walnut Board. p. 4
Littell, R.C., Milliken, G.A., Stroup, W.W. & Wolfinger, R.D. 1996 SAS system for mixed models. SAS Institute Inc., Cary, NC
McGranahan, G.H. & Catlin, P.B. 1987 Juglans rootstocks, p. 411–450. In: Rom, R.C. and R.F. Carlson (eds.). Rootstocks for fruit crops. John Wiley and Sons, New York, NY
McGranahan, G.H. & Leslie, C.A. 1990 Walnuts (Juglans), p. 907–951. In: Moore, J.N. and J.R. Ballington (eds.). Genetic resources of fruit and nut crops. Vol. 2. Acta Hort., Wageningen, The Netherlands
Mircetich, S.M., Browne, G.T., Matheron, M.E. & Teviotdale, B.L. 1998 Armillaria and Phytophthora root and crown rot diseases, Walnut Production Manual. p. 221–232
Redfern, D.B. & Filip, G.M. 1991 Inoculum and infection, p. 48–61. In: Shaw, C.G. III and G.A. Kile (eds.). Armillaria root disease. U.S. Department of Agriculture Forest Service Agriculture Handbook No. 691, Washington, DC
Reil, W. 1997 Walnut rootstock evaluation for resistance to Armillaria root rot, Walnut Research Reports 1997. Walnut Marketing Board. p. 109–113
Teviotdale, B.L. & Sibbett, S. 1999 Armillaria root rot: Role of site preparation and rootstock susceptibility on survival of replanted walnut trees, Walnut Research Reports. California Walnut Board. p. 373–375
Westfall, P.H., Tobias, R.D., Rom, D., Wolfinger, R.D. & Hochberg, Y. 1999 Multiple comparisons and multiple tests. SAS Institute Inc., Cary, NC