Disease background
Armillaria root disease is caused by the fungus Armillaria mellea (Fig. 1), which decomposes the woody roots of trees.  Such destruction to the root system inhibits water and nutrient uptake from the soil, significantly reduces crop growth and yield, and eventually kills infected trees.  Symptoms include stunted shoots, wilting, dwarfed foliage, premature defoliation, and dwarfed fruit (Fig. 2).  A landscape characteristic is disease centers -- localized areas of dead and dying trees (Fig. 3).  Fungicides are of little utility because decomposition of the root crown disrupts their movement through the vascular tissue.  The pathogen also escapes fumigation in deep soil and can persist 10+ years in residual roots.

Fig. 1. Mushrooms of Armillaria mellea form in winter, typically with the first heavy rains in early December. They do not form every year, however, and so they are not the best diagnostic feature of Armillaria root disease.

Fig. 3. This disease center in a peach orchard, same as in Fig. 2, includes a number of dead trees at its margin (sawed off at the trunk), but it is mostly bare. Because the pathogen survives for years on partially-decayed tree roots, replants tend to become infected unless a backhoe is used before planting to retrieve such sources of inoculum buried in the soil.

Fig. 2. Canopy symptoms of Armillaria root disease, shown on a symptomatic peach tree second from left, include stunted shoots, yellowed leaves, and stunted fruit with declining yields over time. Walnut growers: you don’t have it that bad – peach and almond are much more susceptible. Indeed, peach and almond rootstocks with a peach background (e.g., Nemaguard) tend to be susceptible relative to Prunus rootstocks with a plum background (e.g., Marianna 2624). This orchard in Ceres, California was established on Nemaguard rootstock, which we use as our susceptible control in our screening experiment. Notice the three dead trees to the right of the symptomatic tree and the large gap to their right.

Screening efforts
Armillaria screening is notoriously difficult in field and greenhouse experiments.  Greenhouse trials require 2-3 years, infection is hit-and-miss, and infected plants rarely develop symptoms.  Meanwhile there has never been a field test of walnut rootstocks with controlled inoculations. There has been only one properly-designed field test of Prunus rootstocks, but it took 10 years to complete.

Our rapid screening procedure relies on rooted cuttings in tissue culture (Fig. 4).  The vegetative stage of the fungus (mycelium, Fig. 5), is grown in liquid culture for 7 days (Fig. 6).  The culture is then homogenized with a hand-held blender into many small fragments, which are quantified and adjusted to the same concentration across all strains used in the experiment.  The pathogen is inoculated to the surface of the tissue culture medium and grows to colonize the entire medium in two weeks.  Plants are incubated for two months, during which time dead plants are tallied.  Mortality is our measure of resistance; rootstocks with the highest % mortality are the most susceptible.

Fig. 4. Walnut plantlets growing in tissue culture. Inoculation was conducted in 7.7 cm × 7.7 cm × 9.7 cm vessels.

Fig. 5. These fungal filaments, technically known as ‘mycelium’, are the infectious stage of the fungus.

Fig. 6. Liquid cultures of Armillaria mellea (shown here before being blended, i.e. homogenized) are the inoculum used in our screening experiment. It takes only 7 days to produce these cultures.


Results to date
We screened six clonally propagated Paradox walnut rootstocks (AX1, Px1, RR4 11A, RX1, Vlach, VX211) using three Armillaria mellea isolates, along with Northern California black walnut rootstock W17, Persian/English walnut cultivar ‘Chandler’, and Chinese wingnut.  Percent mortality ranged from 9% for the most resistant Paradox rootstock, AX1, to 70% for the most susceptible, VX211 (Figs. 7, 8); this finding was consistent across three isolates of A. mellea and in three replicate experiments.  Chandler, W17, and wingnut had high susceptibility (61%, 69%, and 87% mortality, respectively).

We screened six Prunus hybrids used for peach and almond rootstocks (Hansen 536, Krymsk 1, Krymsk 86, Lovell, Marianna 2624, Nemaguard) using two Armillaria mellea isolates and one Armillaria tabescens isolate (Fig. 9).  Both isolates of Armillaria mellea were equally virulent, while the isolate of Armillaria tabescens was less virulent in all rootstocks.  Marianna 2624 and Nemaguard were included as resistant and susceptible controls, respectively, based on field observations, and accordingly Marianna 2624 had 46% mortality at 2 months post-inoculation versus 76% for Nemaguard.  Krymsk 1 (Fig. 10) and particularly Krymsk 86 (aka Kuban 86) compared favorably to Marianna 2624, Lovell was similar in susceptibility to Nemaguard, but Hansen 536 was the most susceptible.

HORTSCIENCE 48(1):68–72. 2013.

Fig. 7. Results of the walnut screening for Armillaria resistance. Starting from the left: of the six Paradox rootstocks, AX1 was most resistant and VX211 was least. Chandler English walnut, W17 black walnut, and WN×W Chinese wingnut were quite susceptible.

Fig. 9. Krymsk 1 Prunus rootstock under different inoculation treatments. The center two plantlets have Armillaria mellea strains collected from two different infected California orchards. These strains were equally virulent across all six rootstocks and all three trials. On the right, Armillaria tabescens, from an infected orchard in South Carolina, was less virulent. On the left is the non-inoculated control.

Fig. 10. Krymsk 1 and Marianna 2624 Prunus rootstocks had similar mortality two months after Armillaria mellea inoculation. Left to right: Marianna 2624 non-inoculated and inoculated, Krymsk 1 non-inoculated and inoculated.

Fig. 8. Paradox rootstocks with the highest levels of resistance (i.e. lowest levels of mortality) to Armillaria mellea. Images are representative of a subset of four Paradox rootstocks (AX1, Px1, RX1, and Vlach) at one and two months post-inoculation during the first of three replicate experiments. The pathogen is visible on the surface of the tissue-culture medium as white- to tan-colored aerial mycelium.