3.7.97
DIFFERENTIAL RESISTANCE/SUSCEPTIBILITY RESPONSES IN CALLUS CULTURES OF QUERCUS SUBER TO PHYTOPHTHORA CINNAMOMI

B MONTEIROI and G CALDEIRA

UA, Dept. de Biologia, Campus de Santiago, 3810 Aveiro, Portugal

Background and objectives
Cork oak (Quercus suber) is an important forest tree with great economical interest for Portugal. In 1993, Brasier et al. [1] reported the evidence for Phytophthora cinnamomi (Pc) involvement in Iberian oak decline. The effects of different concentrations of plant growth regulators on resistance/susceptibility responses of cork oak tissue cultures to Pc were examined. With 0.01 and 0.1 mg/l of 6-benzylaminopurine (BAP) and 0.5 and 1.5 mg/l of a-naphthaleneacetic acid (NAA), tissues appeared extensively colonized in response to the zoospores of the fungus. The same tissue, growing in media with 1.0 mg/l of BAP and 2.0 mg/l of NAA were only slightly colonized. In this system it is possible to study resistance or susceptibility without changing the host or fungal genotype. Resistance to Pc appears to be regulated by physiological and biochemical mechanisms.

Materials and methods
To initiate in vitro cultures, plant material was subject to superficial sterilization [tap water passage + 1 min. in ethanol 96 + 20 min in disinfecting solution (20% commercial bleach with drops of Teepol) + wash 3 times in sterilized demineralized water]. Portions of internodal segments and petioles were induced to form callus in CTM {callus tissue medium: macro- and micronutrients of modified medium of Gressof and Doy + Fossard and Lee vitamins + peptone 0.5 mg/l + myo-inositol 0, 1 mg/l [2] + BAP 0.1 mg/l + NAA 1.0 mg/l}. The subcultures were done every 4 weeks to CTM. Cultures were maintained always under obscurity in a controlled room (temperature 241C and 16 h light).

In order to evaluate the contribution of the growth regulators to the resistance mechanisms towards the fungus, different combinations of BAP (0.01, 0.1 and 1.0 mg/l) and NAA (0.5, 1.0 and 2.0 mg/l) were tried. Calli were transferred to this media, allowed to grow for 4 weeks, and then infected with Pc zoospore suspensions. Pc (H1000 and IMI 077375 strains) grew in PDA (Difco) or V8 at pH 6-6.5 [3] at 241C. To produce zoospores the modified Gees and Coffey method was used [4]. The soil extract was substituted by modified mineral solution of Chen and Zentemyer [5] to reduce problems related to contamination.

A disc (3 mm diameter) of sterile filter paper was placed on the center of the callus colony and was inoculated with 10 pL of the zoospore suspension. In the controls the zoospore suspension was substituted by sterile distilled water.

Results and conclusions
Differences between the two strains' invasion seemed not to be significant. There was a colour change of the tissues from light-yellow to dark-brown in calli infected by zoospore suspension. A brown halo developed on the agar surface in contact with the callus. In the assayed media differential colonization rates were recorded. The invasion was much reduced in tissues growing with the higher concentrations of BAP and NAA (1.0:2.0 mg/l, respectively). In this medium, after 140 h of incubation, aerial mycelium dispersion was 1.5 cm diameter, while in media containing lower concentrations of fito-hormones (0.01:0.5 mg/l and 0.1:1.0 mg/l) was over 4 cm diameter. The capacity of the fungus to invade the callus decreased with the rise of the concentration both of BAP and NAA. Under different hormonal regimes the callus appeared similar in morphology and texture, so the differential colonization rates could not be explained in terms of morphology. Resistance to Pc appears to be regulated by physiological and biochemical mechanisms.

References
1. Brasier CM, Robredo F, Ferraz JFP, 1993. Plant Pathology 42, 140-45.
2. Gomes L, 1989. Tese de Mestrado., Dept. de Biologia Vegetal, FCL.
3. Miller PM, 1955. Phytopathology 55, 461-62.
4. Chambers SM, Hardham AR, Scott ES, 1995. Mycological Research 99, 1281-88.
5. Dearnaley JDW, Maleszka J, Hardham AR, 1996. Mycological Research 100, 39-48.