Factors Affecting Cytospora Canker Occurrence on Aspen

Duration of wound susceptibility

Three experiments were performed to determine how long wounds on trees remain susceptible to infection under drought stress. One experiment (I) was conducted from September through October, and the other experiments were conducted from February through March (II) and from April through May (III). Seed-propagated, 1.5 m to 2.0 m tall, approximately 2.5 cm diameter, 4-to 5-year-old aspen trees were grown in 19 L black plastic pots in a mixture of 50% topsoil, 25% sand, 20% peat moss, and 5% composted wood chips and cow manure. Trees were maintained at 21 °C to 30°C in a greenhouse for the duration of the experiments. Half of the trees were watered to runoff daily. Stressed trees in experiment I were given 500 ml_ of water per week. In experiments II and III, stressed trees were given approximately 250 ml_ water, rather than the 500 mL per week, when they appeared wilted or when water potential (WP) measured less than -2.0 MPa, so that the plants’ water potential would be more stable. The differential in WP between treatments was maintained from 1 week prior to wounding until conclusion of the experiments. Predawn WP of a leaf from each tree was measured with a pressure bomb (PMS Instrument Company, Corvallis, Oregon). Measurements were made twice weekly in experiment I and on alternate days in experiments II and III.

The C. chrysosperma isolate was obtained from P. deltoides growing in Fort Collins, Colorado, and was maintained on potato-dextrose agar (PDA) plates at 24°C to 30°C in dark. Mycelium on agar discs (8 mm in diameter) was cut aseptically from 7-day-old cultures and used as the inoculum source.

Wounds were made on day 0 at 15 cm intervals along the stems after bark was wiped with 70% ethanol. Longitudinal, 16 mm long wounds extending just into the xylem were made with a disinfested cold chisel on all trees. In experiment I, 10 stressed and 10 nonstressed trees were each wounded 6 times on day 0. Each tree was then inoculated at one of the wounds at 0, 2, or 7 days after wounding. Agar disks were applied as controls to the other 3 wounds at the same time as inoculations were made. In experiment II, 22 stressed and 22 nonstressed trees were inoculated at 2, 4, 5, and 6 days after wounding to determine the duration of wound susceptibility suggested to be between 2 and 7 days in experiment I. Because the duration of wound susceptibility was apparently longer in this spring experiment, a third experiment was initiated. In experiment III, 12 stressed and 12 nonstressed trees were inoculated on days 0, 2, 7, 10, or 14 days after wounding. After inoculation, wounds were wrapped with parafilm for 2 days. Locations of wounds to be inoculated on a specific day were randomized. To determine if C. chrysosperma was present in the inoculated wounds, samples were taken at all wound margins 3 weeks after inoculation.

Canker length, defined by bark discoloration, was measured 2 weeks after inoculation (Table 2). For each experiment, mean canker length of stressed versus nonstressed trees was compared by a Student’s t-test.

Cytospora in/on bark

To determine if C. chrysosperma is a bark inhabitant, 29 asymptomatic trees in 3 natural aspen stands in the Red Feather Lakes area of Roosevelt National Forest in northern Colorado were sampled monthly from May 1986 through April 1987. One stand was about 1.6 km from the other two, which were about 100 m apart. At each sampling time, bark was swabbed with a 1:9 solution of sodium hypochlorite:water followed by swabbing with 70% ethanol and allowed to dry. One 9 mm diameter bark sample (phloem only) per tree was removed immediately with a sterile cork borer and placed bark side up on PDA. This was a test of Cytospora presence in bark, since we assumed Cytospora outgrowth, if present, would originate from beneath the bark surface because these samples were surface disinfested. A second bark sample, not surface disinfested, was removed from an area 2 cm to 3 cm below the first sample site. This sample was placed bark side down on PDA. This was a test of the presence of Cytospora on the bark, since we assumed Cytospora outgrowth would originate from the bark surface because these samples were not surface disinfested. All samples were incubated at 24°C to 30°C in a lab for 2 to 14 days.

Virulence of Cytospora

Two experiments were conducted to assess the virulence of C. chrysosperma isolates on excised aspen branches. Aspen branches approximately 1.5 cm in diameter at the base were excised from trees from several groves in the Red Feather Lakes area. Branches were placed in plastic bags and transported on ice to the laboratory. Branches were placed upright in plastic bags with their bases in distilled water and refrigerated until they stopped gaining weight. In experiment I, we assessed the relative virulence of 8 isolates, 1 isolate obtained from each of 8 Populus and Salix species (Table 1). One wound was inflicted longitudinally with a cold chisel on 15 to 22 branches per isolate at sites previously wiped with 70% ethanol. Inoculum consisted of mycelium on agar disks (8 mm diameter) cut aseptically from 1 -week-old PDA cultures. After inoculation, wounds were wrapped with parafilm for 3 days. Inoculated branches were placed upright with their bases in distilled water in translucent plastic bags and incubated at 24°C to 30°C in a lab. Canker length was measured 2 or 3 weeks after inoculation. For experiment II, we compared 5 C. chrysosperma isolates obtained from 5 different P. tremuloides trees by inoculating 11 aspen branches per isolate for a total of 55 branches. Analysis of variance of mean square-root-canker length and LSD (P = 0.05) analyses were utilized.

Duration of wound susceptibility

Duration of wound susceptibility was similar for stressed and nonstressed trees in the fall experiment (I) but not in the spring experiments (II and III). In experiment I, susceptibility lasted between 2 and 7 days (Table 2). Cankers developed on all wounds inoculated at 0 and 2 days. No cankers developed on control wounds. In experiment II, susceptibility of stressed trees lasted longer than susceptibility of nonstressed trees (Table 2). Only a few wounds on nonstressed trees were susceptible 2 days after wounding, whereas wounds on stressed trees remained susceptible through 6 days. Experiment III refined and confirmed the duration that wounds remain susceptible (Table 2). Stressed trees had wounds that were susceptible for 10 to 14 days after wounding, and nonstressed trees had susceptible wounds for only 4 days after wounding. Since there was only one fall experiment, we cannot determine if the duration of wound susceptibility is less in the fall as suggested by our experiment. Mean canker size for stressed trees was significantly larger than canker size on nonstressed trees on all experiments (Table 2). Mean WP over the entire experiment for stressed and nonstressed trees, respectively, in these 3 experiments were:

• Experiment I: –1.57 MPa, and –0.32 MPa

• Experiment II: –1.50 MPa, and –0.30 MPa

• Experiment III: –1.46 MPa, and –0.47 MPa

Cytospora in/on bark

Cytospora was isolated 38 times from the 349 samples assayed for the pathogen on the bark (Table 3). Thirty-six of the isolates were obtained from samples collected from June through November. One isolate was collected in May and one in December. Cytospora was never isolated from the 349 samples assayed for presence of the pathogen in the bark.

Virulence of Cytospora

We found that isolates of Cytospora from different hosts caused significantly different canker sizes on inoculated aspen branches, as did different isolates from different aspen trees (Tables 4 and 5). Interestingly, isolates 11 and 13, both collected in the Red Feather Lakes area, caused significantly different average canker sizes.