In vitro expression of resistance responses to ...

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In vitro expression of resistance responses to Seiridium species in micropropagated shoots of Cupressus sernpervirens and Charnaecyparis

K.A. Spanos, A. Pirrie, and S. Woodward

Abstract: Wounded and nonwounded micropropagated shoots of C~tpressussernpervirens and Chnrnnecypnris lnwsoninnn were inoculated in vitro with the canker-causing pathogens Seiridirlrn cardinale (Wag.) Sutton & Gibson, Seiridium crcpressi (Guba) Boeswinkel and Seiridiurrz unicorne (Cke & Ell.) Sutton. Seiridium cnrrlinale was significantly more pathogenic on Cupressus sernpervirerzs than on Charnnecypnris ln~vsoninrza(Murr.) Parlatore, irrespective of the presence of wounds on the shoots. On wounded shoots, both S. crtpressi and S. urriconze caused significantly larger lesions on Charnaecyparis ln~vsorriarrnthan on Crtpressus sernpervirens by 20 days after inoculation. Superficial wounding of shoots prior to inoculation caused a significant increase in the lengths of lesions and numbers of shoots girdled by the pathogens on both hosts. These results broadly correlate with known virulence of the three pathogens on these two host tree species in field and glasshouse tests. Using histological methods, penetration of fungal hyphae through stomatal pores of both shoots and leaves into the substomatal cavity and the mesophyll space was observed. Penetration directly through the cuticle was also seen. Defence-related responses, including accumulation of oxidized polyphenolic compounds and deposition of lignin and suberin in cell walls, were detected in inoculated tissues. These responses occurred predominantly in the epidermis, including stomatal guard cells, and the hypodermis and were particularly marked in Chnrnnecyparis lmvsorriana inoculated with S. cardinale. 'The possible utility of these methods in the study and detection of host genotypes resistant to Seiridi~trnspp. is discussed.

Key worrls: Seiridiuml, Cupressus, Charnnecyparis, micropropagation, in vitro inoculation, defence.

RCsumC : Les auteurs ont inoculk, in vitro, des tiges blessCes ou non blessCes de Cupressus serrrpervirens et de Clrarnnecypan's lawsoniarm avec des champignons pathogknes causant des chancres, soit les Seiridirtm cardinale ((Wag.) Sutton & Gibson), Seiridiurn cltpressi (Guba) Boeswinkel) et Seirirlrtrn urricorrre (Cke & Ell.) Sutton. Le S. cnrrlirrnle est significativement plus pathogkne sur le Cupressrts serrrpervirerrs L. que sur le Charnnecypnris lavvsoniarrn (Murr.) Parlatore, indkpendamment de la presence de blessures sur les tiges. Sur les tiges blessCes, le S. crtpressi et le S. rtnicorne causent des Itsions significativement plus grandes sur le Charnaecyparis ln,vsoniana que sur le C~tpressrts sernpervirens, 2 0 jours aprks I'inoculation. Une blessure superficielle des tiges avant I'inoculation entraine une augmentation de la longueur des ICsions et du nombre de tiges CtranglCes par le champignon pathogkne, sur les deux hbtes. Ces resultats correspondent en gCnCral aux virulences des trois pathogknes telles qu'on les observe sur ces deux hbtes lors d'essais aux champs ou en serre. A l'aide de mCthodes histologiques, les auteurs ont observC la pknetration de I'hyphe fongique par les ostioles des stomates des feuilles aussi bien que des tiges, le passage dans les chambres sous-stomatales et dans les espaces du mCsophylle. Ils ont Cgalement observe des pCnCtrations directes B travers la cuticule. Dans les tissus inoculCs, les auteurs ont observC des reactions associCes B la dCfense, incluant I'accumulation de substances phenoliques oxydCes et la deposition de lignine et de subkrine dans les parois cellulaires. Ces rCactions surviennent surtout dans I'Cpiderme, incluant les cellules de garde des stomates, et dans I'hypoderme; elles sont particulikrement fortes chez le Charnaecyparis la~tsoninrrninoculC avec le S. cnrdirrale. Les auteurs discutent de I'utilisation possible de ces methodes pour I'Ctude et la dCtection de genotypes hbtes rCsitants aux Seiridiurn spp.

Mots cle's : Seiridirtm, Cupressus, Charnnecyparis, micropropagation, inoculation in vitro, defense. [Traduit par la ridaction]

Received September 13, 1996.

Introduction

K.A. Spanos,' A. Pirrie, and S. W o o d w a ~ d .Department ~ of Forestry, University of Aberdeen, MacRobert Building, 581 King Street, Aberdeen AB24 5UA, Scotland, UK.

Seiridium species cause serious canker diseases on members of the Cupressaceae. Seiridium cardinale (Wag.) Sutton & Gibson, first reported from Cupressus macrocarpa Hartweg plantations in California in the 1920s (Wagener 1928), is the most serious pathogen, resulting in dieback of susceptible hosts. Dissemination of Cupressus macrocarpa through many regions of the world is considered responsible for the

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Present address: Forest Research Institute, 57006 - Vasilika, Thessaloniki, Greece. Author to whom all correspondence should be addressed. e-mail: [email protected]

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widespread incidence of S. cardinale in North and South America, Europe, Australia, and New Zealand (Panconesi 1990). Two further species, Seiridium cupressi (Guba) Boeswinkel and Seiridium unicorne (Cke & Ell.) Sutton, also cause serious canker diseases on Cupressaceae (Panconesi 1990). Despite the destructive nature of these pathogens, great variation in susceptibility exists both between and within species of Cupressus (Andreoli 1979; Grasso and Ponchet 1979; Raddi 1979). Effective exploitation of this resistance would enable the use of more productive species of Cupressus in areas where a high level of the disease currently exists. In vitro screening procedures have great potential in forest pathology and forest genetics, offering several advantages over conventional glasshouse and field resistance testing. The use of such systems for the study of resistance to pathogens in forest trees has received considerable attention over the last 15 years (Amerson and Mott 1982; Diner and Karnosky 1987; Ostry and Skilling 1992). Tissue culture methods have proven particularly valuable in the study of rust pathogens of conifers (Diner and Mott 1982; Diner et al. 1984), and the value of these techniques in the study of necrotrophic pathogens has also been demonstrated (Abdul Rahman et al. 1987; Bronson et al. 1992; Nsolomo and Woodward 1994; Ostry et al. 1990). Such methods, however, have received only limited attention in the SeiridiumCupressus interaction (Spanos and Woodward 1996; Tonon 1994; Tonon et al. 1995). Various in vitro methods, including cell suspensions (Lesney 1989), callus (McComb et al. 1987; Hrib and Rypacek 1981; Diner et al. 1984; Woodward and Pearce 1988), embryos (Diner and Mott 1982; Gray and Amerson 1985), and micropropagated shoots (Jacobi 1982; Abdul Rahman et al. 1987; Ostry et al. 1990), have been used in the study of diseases of forest trees. Resistance responses, such as rapid tissue necrosis, hypertrophy and hyperplasia, wall appositions, and development of periderm, which correlate well with reactions observed in the field, could be used to evaluate resistance in individual genets or seed lines using much less time and space than conventional methods (Diner and Mott 1982; Gray and Amerson 1985; Abdul Rahman et al. 1987). Traditional field screening tests for resistance to Seiridium canker of Cupressus spp. take 3-4 years to complete, depending on weather conditions. An in vitro system, therefore, offers the potential for rapid screening for resistance to this pathogen. Resistant genotypes identified by in vitro screening could then be outplanted to high risk sites for verification of field resistance. In addition, in vitro challenge of tissues of selected host genotypes may be useful in the study of the basic processes of resistance and pathogenesis. This paper reports an in vitro method for detecting resistance responses in Cupressus sempewirens and Chamaecyparis lawsoniana inoculated with S. cardinale, S. cupressi, and S. unicorne.

Materials and methods Plant material Eighteen-month-old seedlings of Cupressus setnperviretu and Chatnaecyparis lawsoniana were maintained under glasshouse conditions, with no overhead watering, for 14 days prior to removal of material

for culture. Shoot tips 50 mm in length were removed from the top of the crown, placed inside plastic bags, and transferred immediately to the laboratory for surface sterilization. Explants were washed for 10 min in running tap water, rinsed in distilled water for 15 min, and sterilized by immersion for 10 min in 30% v/v H 2 0 2containing 0.025 % Tween 20 as a wetting agent, followed by 20 min in 20% v/v commercial bleach (Domestos; 6 % NaOCI) in tap water. Explants were then rinsed in five changes of sterile distilled water, aseptically trimmed to 20 mm in length, and inserted into 10 mL culture medium in 30-mL glass jars.

Culture conditions Murashige and Skoog (1962) modified medium (MSM; Sigma M0153), 2.2 g . L - ' in distilled water, supplemented with (per litre) 2 mg glycine, 100 mg inositol, 0.5 mg nicotinic acid, 0.5 mg pyridoxine HCI, 0.1 mg thiamine HC1, and 30 g sucrose, was used for plant culture. Bacteriological agar (Oxoid; 0.6%)was added and the pH adjusted to -5.7 using a few drops of 1 M HCI or I M NaOH before autoclaving at 105 kPa for 20 min. Explants were initially cultured on MSM with 3 % sucrose for 10 days for acclimation and elimination of contaminated cultures. For shoot multiplication, contaminant-free explants were transferred to MSM containing 3% sucrose and benzylaminopurine (BA; 0.0- 1.0 mg . L - ' ) for 14 days for axillary shoot proliferation and development. Prior to rooting, micropropagated shoots were conditioned on MSM with 2 % sucrose, but without growth regulators, for 28 days. Cultures were maintained in the growth room at a constant 25 f 2 ° C (mean f SD) with a 16 h light : 8 h dark photoperiod under a light intensity of 19-23 pmol . m-' . s-', provided by Phillips MCFE 40W/29 fluorescent tubes.

Fungal material ~ ~ c e l i isolates al of S. cnrdinnle, S. cupressi, and S. utlicor-ne, obtained from the culture collection of Dr. S.G. Xenopoulos, Forest Research Institute of Athens, Greece, were maintained on 3 % malt extract agar (Oxoid) at 25 f 2°C. These fungi were isolated from infected Cupressus sempetvirens in Italy (ATCC 38654), Kos (Greece, "grey" isolate), and Portugal (SP AI86), respectively (Xenopoulos 199 1).

In vitro inoculations Rooted shoots of Cupressus sempervirens and Cllatr~aecyparis lawsoniatza, 50-80 mm in length growing in MSM without growth regulators, were inoculated with 2 mm diameter plugs of S. cardinale, S. cupressi, and S. unicorne taken from the edge of actively growing 1-week-old colonies growing on 3 % malt agar. Inocula were inverted onto host tissues at the midpoint between the shoot apex and the base, with the hyphae pressed closely against the main stem of the shoot. Half of the shoots from each species were wounded by removing the primary leaves to expose a sub-epidermal surface wound of approximately 1 x 2 mm. Inoculated shoots were returned to the growth room and completely randomized on the illuminated shelves. Shoots were examined for development of symptoms 10 and 2 0 days after inoculation. Lesion length (length of the dead or discoloured area on the stem) and stem girdling (development of necrosis all around the stem) were recorded. Tissues were then fixed for histological examination.

Experimental design A factorial randomized design with the 2 tree species, 3 Seiridium species, and 2 inoculation treatments (wounded and unwounded) was used, giving a total number of 12 treatments, with 2 0 replicate shoots per treatment. Experiments were repeated at least twice. Results were analysed using ANOVA and chi-square (X2) tests. @ 1997 NRC Canada

Spanos et al. Table 1. Lengths of lesions caused by Seiridium cardinale, S. cupressi, and S. unicorne on micropropagated shoots of Cupressus sempervirens and Charnaecyparis larvsotziana inoculated in virro.


Lesion length (mm) Host

Treatment*

Cupressus setnpervirens

NW W

Chatnaecyparis lawsotziana

NW W

Days after inoculation 10 20 10 20 10 20 10 20

Seiridiutn cardinale

Seiridiurn cupressi

Seiridiutn unicortze

2.50f 0.22d 2.60f 0.26d 10.65f0.61ab 13.65f 0.65hi 3.25f0.34m 3 . 1 5 f 0.33tn 12.40f 0 . 5 7 ~ 9.70f 0 . 4 6 6 ~ 9 . 7 0 f 0 . 5 3 6 ~ 15.15 k0.54h 9.95 k0.53j 19.30k0.60g 8.45_+0.58c 3.95 k 0 . 5 1 ~ 3.65f0.46d 7.15 *0.93k 6.75f0.88k 10.15 k0.97j 9.40f0.556~ 9.55f 0.506~ 8 . 6 5 f 0 . 4 9 ~ 18.60+0.91g 13.40f0.88hi 11.90f0.82ij

SE of 20 replicate shoots per treatment. Means followed by the same Note: Values are the means letter do not differ significantly ( P < 0.01; LSD test). *NW, nonwounded shoots; W, shoots wounded prior to inoculation.

Least significant difference (LSD) was used for comparison of means in the treatments.

Histology of shoots inoculated in vitro Inoculated shoots were fixed in formalin - acetic acid - alcohol for 24 h (Jensen 1962), dehydrated in t-butyl alcohol, and embedded in paraffin wax (melting point 56-57°C). Sections (1015 pm) were cut using a sledge microtome and mounted on glass slides. Following removal of wax in 1,1,1-trichloroethylene,sections were rehydrated in an ethanol-water series before staining. Sections were stained with aniline blue, as a general stain, phloroglucinol HCI, as a stain for ligninlike materials, or Sudan IV, as a stain for lipids and lipid polymers (Jensen 1962). Light and UV microscopy was carried out using a Carl Zeiss Jenamed 2 fluorescence microscope, equipped with an HBO 50-W mercury burner, with 340- to 380-nm excitation and 410- and 450-nm barrier filters.

Results Symptom development on micropropagated shoots inoculated with Seiridium spp. Inoculated shoots were examined in vitro under a dissecting microscope 1, 10, and 20 days after inoculation. Abundant regrowth of hyphae was observed 24 h after inoculation, with many hyphal tips touching the stem and needle surface around the inoculation point. Tissue discoloration was evident, particularly on wounded shoots, and increased between 10 and 20 days after inoculation. Greater growth of aerial hyphae was observed in dual cultures of plant material with S. cardinale than S. cupressi or S. unicorne. Acervuli of S. cardinale were observed on most shoots of Cupressus sempervirens by 10 days after inoculation and were abundant on wounded shoots of this host species but rarely formed on Chamaecyparis lawsoniana. Lesion sizes and percentage stem girdling following inoculation are shown in Table 1 and Figs. 1 and 2, respectively. In terms of lesion size, S. cardinale was significantly more pathogenic on shoots of Cupressus sempervirens than on Chamaecyparis lawsoniana (P < 0.01). These differences were apparent on both nonwounded and wounded shoots. Seiridium cupressi caused significantly larger lesions

(P < 0.01) on nonwounded shoots of Chamaecyparis lawsoniana than on Cupressus sempervirens at 10 and 20 days after inoculation. In wounded shoots, lesions on Chamaecyparis lawsoniana were similar in size at 10 days postinoculation but were significantly larger (P < 0.01) by day 20. Seiridium unicorne also caused significantly larger lesions (P < 0.01) on both wounded and nonwounded shoots of Chamaecyparis lawsoniana than on Cupressus sempervirens, although this difference was not apparent until 20 days after inoculation. Compared with nonwounded shoots, wounded shoots of both Cupressus sempervirens and Chamaecyparis lawsoniana developed significantly larger lesions (P < 0.01) following inoculation with all three Seiridium species. These increases were particularly marked on shoots inoculated with S. cupressi and S. unicorne, where lesion sizes increased by three- to five-fold with wounding. Lesion sizes increased significantly between 10 and 20 days after inoculation (P < 0.01). In nonwounded shoots, S. cardinale caused significantly greater levels of girdling than S. cupressi or S. unicorne on both host species (Figs. 1 and 2). The percentage numbers of shoots of Cupressus sempervirens with total stem girdling 10 days after inoculation with S. cupressi or S. unicorne increased from zero in nonwounded to 70% in wounded shoots. With S. cardinale inoculation, the increase was from 65 % girdling in nonwounded to 85 % in wounded shoots. By 20 days after inoculation, 100% of Chamaecyparis lawsoniana shoots were girdled by both S. cupressi and S. unicorne, compared with 95 and 90%, respectively, in Cupressus sempervirens. Histology of inoculated shoots Transverse sections of infected leaves of Cupressus sempervirens, taken from immediately adjacent to the lesion showed abundant hyphae in the mesophyll, with fewer hyphae penetrating the vascular cylinder (Fig. 3). Destruction of the mesophyll tissues was apparent in both Cupressus sempervirens and Chamaecyparis lawsoniana by 10 days after inoculation, when compared with sections of control shoots (Fig. 4). Examination revealed hyphal penetration of all Seiridium spp. into the mesophyll through stomata1 pores O 1997 NRC Canada

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Fig. 1. Stem girdling on micropropagated shoots of Cupressus sempervirens inoculated in vitro with Seiridiurn cardinale (w), S. cupressi (a), and S. unicorne (a). Percentage of girdled shoots was recorded 10 and 20 days after inoculation. NW, nonwounded shoots; W, wounded shoots. n.s., not significant, P > 0.05; ***, P < 0.001. Values above bars are the absolute values on the y-axis.

Fig. 2. Stem girdling on micropropagated shoots of Chamaecyparis lawsoniana inoculated in vitro with Seiridium cardinale (w), S. cupressi (a), and S. unicorne (o). Percentage of girdled shoots was recorded 10 and 20 days after inoculation. NW, nonwounded shoots; W, wounded shoots. n.s., not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001. Values above bars are the absolute values on the y-axis.

(Fig. 3), or in a few observations directly through the cuticle. Penetration to the mesophyll space and substomatal cavity was commonly seen in sections from both host tree species. Accumulation of oxidized polyphenolic compounds in cell walls, defined by the presence of an orange-brown colour, was observed in cells of the infected epidermis and mesophyll, and also in the vascular cylinder (Figs. 5 and 6). These deposits were not seen in leaves of uninoculated shoots (Fig. 4). Lignified areas within cell walls stained red with phloroglucinol HCl, and, in the absence of staining, displayed marked autofluorescence when viewed under UV illumination. Lignification was observed in stomata1 guard cells,

in the epidermis, and in the hypodermis immediately surrounding the stomata where penetration occurred. A necrotic reaction was also observed in mesophyll cells of Chamaecyparis lawsoniana. Formation of a strong reaction to infection with Seiridium spp., and particularly to S. cardinale, was observed only in leaves and shoots of Chamaecyparis lawsoniana. In this host species, a ligno-suberized zone, detected by autofluorescence of unstained sections under UV illumination with a 410-nm barrier filter and quenching of autofluorescence following staining with phloroglucinol HC1, always formed in the epidermis and mesophyll tissues immediately surround@ 1997 NRC Canada


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Figs. 3-6. Light photomicrographs of transverse sections from micropropagated shoots of Cupressus sernpervirens (Fig. 3) and Chamaecyparis lawsoniana (Figs. 4-6). Fig. 3. Transverse section of Cupressus sempervirens leaf 10 days after inoculation with Seiridium cardinale. Note the necrosis of mesophyll (M) and vascular cylinder (VC) tissues. Penetration of a hypha through a stoma can be seen (arrow). Hyphae are also visible in the substomatal cavity and in the mesophyll spaces (arrow heads). Stain: toluidine blue. Scale bar = 40 pm. Fig. 4. Healthy leaf of Chamaecyparis lawsoniana. Stain: toluidine blue. RC, resin canal. Scale bar = 100 pm. Fig. 5. Chamaecyparis lawsoniana leaf 10 days after inoculation with Seiridium cardinale. Abundant hyphae (arrow heads) are present on the epidermis and within the mesophyll (M) space. Arrows indicate browning of cell walls in the epidermis, suggesting deposition of polyphenolic material. Stain: toluidine blue. Scale bar = 20 pm. Fig. 6. Twenty days after inoculation, extensive necrosis, along with deposition of ligninlike materials, (arrows) has developed in the leaf epidermis and hypodermis of Chamaecyparis lawsoniana, adjacent to fungal inoculum, but hyphae have not penetrated this barrier. No stain. Scale bar = 100 pm.

ing stomata where hyphal penetration had occurred. This response was not observed in Cupressus sempervirens.

Discussion Symptom development following in vitro inoculations of Cupressus sernpervirens and Chamaecyparis lawsoniana with S. cardinale, S. cupressi, and S. unicorne generally reflected the relative pathogenicity of these fungi determined in glasshouse experiments (Spanos 1995) and in field experiments reported by other authors (Beresford and Mulholand 1982; Xenopoulos 1991). Single isolates of each Seiridium species were used in the current study as previous work indicated that there is little within-species variation in virulence for these pathogens (Xenopoulos 1991; Spanos 1995). Seiridium cardinale inoculated shoots of both host tree species showed the most intense symptoms 10 days after inoculation, although overall symptoms were most severe on shoots of Cupressus sempervirens. Ten days after inoculation, S. cupressi and S. unicorne caused similar-sized lesions, but those caused by S. cupressi were larger by 20 days postinoculation. In inoculated wounds in field and glasshouse tests, Chatnaecyparis lawsoniana has a high level of resistance to S. cardinale (Wagener 1939; Strouts 1973; van der Werff 1987;

Spanos 1995) but is more susceptible to S. unicorne (Fuller and Newhook 1954; Gilmour 1966; Spanos 1995). For both lesion length and percentage stems girdled by the pathogens, the results presented here suggest that, following wounding, Chamaecyparis lawsoniana is more susceptible to S. cupressi than to S. cardinale or S. unicorne. In the absence of wounding, however, S. cardinale inoculation produced the most pronounced symptoms. These results should also be considered in terms of the increase in lesion lengths over the time of the experiments. In wounded shoots of Chamaecyparis lawsoniana, lesions caused by S. cupressi and S. unicorne increased in length markedly between day 10 and 20, whereas the increase on S. cardinale inoculated shoots was relatively small. This apparent decrease in the growth rate of S. cardinale, which is also reflected in the nonwounded shoots, may result from the development in the host of more effective resistance barriers against this pathogen than against S. cupressi and S. unicorne. Correlations between responses of in vitro inoculated conifers to necrotrophic pathogens and known levels of field resistance have also been reported in adventitious shoot cultures of Larix species infected with Gremmeniella abietina (Abdul Rahman et al. 1987) or with Mycosphaerella laricina (Ostry et al. 1990). Moreover, similar positive correlations occur in callus and embryo culture of various coniferous

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Can. J. Bot. Vol. 75, 1997 species, including members of the Cupressaceae, inoculated with necrotrophic pathogens (Abdul Rahman et al. 1987; Spanos and Woodward 1997). Although these methods are not as advanced as those applied to rust pathogens of conifers (Diner and Mott 1982; Diner et al. 1984; Gray and Amerson 1985; Jacobi 1982) where the genetics of host resistance are better understood, their application in resistance breeding programmes could have marked advantages in terms of speed and accuracy, particularly if combined with physiological tests following treatment with toxins (Tonon 1994; Tonon et al. 1995). Histological examination of inoculated tissues of Cupressus sernpervirens and Charnaecyparis lawsoniana indicated that rapid destruction of tissues immediately surrounding the inoculation occurs, with hyphae of all three Seiridiurn species penetrating leaves and unwounded stems via stomata1 apertures. The observation that hyphae may also penetrate directly through the cuticle is unusual, as this infection route is not known from the field. However, the relatively thin nature of the cuticle on tissues maintained in vitro (Abdul Rahman et al. 1987; Ostry and Skilling 1992) may result in a lower resistance to direct penetration by hyphae of pathogenic fungi. Lignification and cell wall thickening responses were observed in both tree species in response to challenge by all three Seiridiurn species. This response was more pronounced than Cupressus sernpervirens, in Charnaecyparis la~~soniana and resulted in the development of a tissue resembling the ligno-suberized barrier seen in stems of Cupressus and Charnaecyparis seedlings inoculated in the glasshouse (Spanos 1995). Lignification and (or) suberization have been reported in other conifer-pathogen interactions in vitro, including callus of Cupressus spp. challenged with S. cardinale (Spanos and Woodward 1997), callus of Picea sitchensis (Bong.) Carr. inoculated with decay fungi (Woodward and Pearce 1988), and cell suspension cultures of Pinus elliotti Engelm. treated with basidiospores of Cronartiurn quercuurn (Berk.) Miyabe ex Shirai f.sp. fusiforrne (Cumm.) Burds. et Snow (Lesney 1989). Deposition of suberin and lignin around infection areas is a common defence reaction in plant tissues following wounding and challenge with potential pathogens (Biggs 1992; Woodward 1992). The speed of development of such barriers could prove a useful measure of the relative resistance of different clones to pathogens, particularly those requiring wounds for entry. Differences between the responses of the two host species in vitro and in field experiments probably resulted from the large differences in conditions between the two growing environments (Ostry and Skilling 1992). Apart from the problem of the thin cuticle, the developmental stages of the plants at the time of inoculation in vitro may differ to those found in the field. The possible influence of plant growth regulators on the responses of cultured plant tissues to fungal challenge must also be taken into account (Harvey et al. 1971; Haberlach et al. 1978; Woodward and Pearce 1988). In using tissue culture techniques to study host-pathogen interactions, it is most important to establish that responses found in vitro correlate with those found under more natural conditions. Only single genotypes of Cupressus sernpervirens and Charnaecyparis lawsoniana were used in this study. Further development of the system is required, therefore, to

assess the extent of intraspecific variation in resistance to Seiridiurn spp. expressed in in vitro inoculated shoots of the Cupressaceae. The results described here, demonstrating broadly similar responses in vitro to those reported in vivo, particularly in Cupressus sernpervirens inoculated with Seiridiurn spp., further emphasize the utility of in vitro systems in the rapid assessment of the disease resistance status of host tree genotypes.

Acknowledgements This work forms part of a doctorate thesis of the senior author. K.A.S. was funded by the Scholarship Foundation of Greece. The technical assistance of Janis Brodie is gratefully acknowledged.

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