7.3 RESULTS 16
8.2.1 Phenolic Compounds
Petals of susceptible and resistant camellia flowers were collected for inoculation and extraction (Table 8. 1 ). For each specimen, four samples of 20 g of petals were weighed out. Three of the samples were inoculated using the airborne inoculation chamber (Section 6.2.2). After inoculation (assessed 2 hourly using cover slips), the petal samples were transferred to a humid chamber and incubated at 20°C. The first sample was removed after 24 h for extraction, the second at 48 h and the third at 72 h. The uninoculated sample was extracted immediately. Four petals of susceptible C. saluenensis x C. reticulata ' Brian' were included in every inoculation to ensure that the C. camelliae ascospores were infective. These petals were transferred to the humid chamber along with the sample petals and incubated until large C. camelliae
lesions were evident. Sample petals may have been infected naturally prior to collection.
8.2. 1 . 1 Extraction, Fractionation and Storage of Samples
The method of Daayf et at ( 1 997) was used for extraction, fractionation and acid hydrolysis of samples. Extraction: 20 mg of petal tissue and 200 ml of acidified 80%
methanol (PH 2.0 with HCl) were placed in prechilled 250 ml conical flasks and placed on ice. Samples were homogenised using a high speed blender (Polytron, Kinematica, GmbH), flushed immediately with nitrogen (-90 sec) and covered with aluminium foil and placed on an orbital shaker at 1 75 rpm at 4°C in darkness. After
48 h, extracts were clarified under suction through moistened filter paper (Whatmann
# 1 , 1 1 cm diameter) in a Buchner funnel. A volume of 20 ml 80% acidified methanol was used to wash the residue. Fractionation: extracts were thrice partitioned against 1 25 ml of light petroleum ether (Ajax Chemicals, UNIV AR) in separating funnels, with the extract and ether shaken vigorously together and allowed to settle (60-90 sec). The methanolic extracts were kept, while the lighter petroleum ether Fraction I extracts were discarded. The methanolic extracts were rotary evapourated under pressure at 38°C to leave 50 ml of extract then partitioned thrice against 30 ml of diethyl ether in separating funnels, with the extract and diethyl ether shaken vigorously together and allowed to settle. The portion in diethyl ether containing free phenolics was kept as Fraction II extracts, while the other portion became the Fraction III extracts containing glycosidically-bound phenolics post-acid hydrolysis. Acid hydrolysis: extracts were diluted with an equal volume of 4N HCI, and placed in conical flasks in a 1 00°C water bath for 90 min. The extracts were filtered under suction through moistened filter paper (Whatmann # 1 , 1 1 cm diameter) in a Buchner funnel. They were then partitioned thrice against 30 ml of diethyl ether (as described above) and the Fraction III extracts retrieved from the diethyl ether portion. Concentration of extracts in vacuo: Fractions II and III were rotary evapourated under pressure at 38°C to a volume of 5- 1 0 ml, then placed in a fumehood for final evapouration to 1 ml. Samples were stored at 4°C in stoppered soda glass specimen tubes.
8.2. 1 .2 Detection of Phenolic Compounds
Samples were spotted onto silica gel ( 1 0-40 J..lm) (Sigma) plates using capillary tubing. Two spots of each sample (one of 20 J..lI and one of 30 Ill) were run on the same plate and each plate had a control spot of 30 J..ll diethyl ether. The spots were placed 2.5 cm from the base of the plate. Dried plates were placed in the saturated
solvent chamber with dichloromethane:n-hexane:methanol (6:4: I v/v) and developed for �20 min. The solvent front was marked on removal from the chamber, and the plate dried for 1 12 -2 h.
The presence or absence of phenolic compounds of each species and sample was assessed using up to six methods:
a) bioassay (conidial suspension of Cladosporium spp.) for detection of
antifungal compounds (Daayf et al 1 997) (Cladosporium spp. was
maintained on Difco PDA. A concentrated conidial suspension was prepared by scrapping the conidia from the surface of the plate and mixed (1 : 1 , v/v) with a 20 gll solution of PDA, then sprayed onto silica gel plates.)
b) visible light (430-790 nm) for anthocyanins, quinones and deeply coloured aurone and chalcone pigments (Seikel 1 962)
c) short-wave ultraviolet light (254 nm) for flavonoid compounds (SeikeI 1 962) d) long-wave ultraviolet light (365 nm) for flavonoid compounds (Seikel 1 962) e) Prussian blue reaction ( 1 % aqueous K2Fe(CN)6 and 1 % FeCh.6H20) for
phenolic compounds of all types (Randerath 1 970; Harbome 1 989)
f)
acid vanillin ( 1 0% vanillin w/v in conc. HCI) for phenols, especially resorcinol and phloroglucinol derivatives, flavans and leucoanthocyanins (Harbome 1989)The Rr (Relative front) value was calculated for each band via the methods b-f, or zone of inhibition (bioassay) and averaged over the two spots per sample. This was a measure of the distance travelled by the compound from the point of origin in a given solvent system and was defined as:
Rr = distance moved
distance moved by solvent front
The Rr value allowed bands from different chromatograph plates, and different seasons, to be compared.
8.2. 1 . 3 Collection and Treatment of Camellia Petals
The experiment was conducted over three camellia flowering seasons (Table 8. 1 ). The first season tested whether the method was suitable for camellialC. camelliae
samples, and whether the (lengthy) preparation process could be duplicated. In the second and third seasons, testing was expanded to cover more susceptible and resistant species. The large number of flowers required for each species (80 g - a very large number of flowers for the small-flowered species), and the time required to process the samples, limited the scale of this experiment.
In Season 1 two samples from each species, consisting of an uninoculated sample and a sample inoculated with ascospores of C. camelliae were incubated at 20°C for
5 d before extraction and tested for Fractions 11 and 11. In Seasons 2 and 3 , four samples were taken from each species: an uninoculated sample extracted immediately (0 h) and three samples inoculated with ascospores of C. camelliae then incubated at 20°C for 24 h, 48 h or 72 h before extraction. They were assessed for Fractions 11 (except Cladosporium spp. bioassay) and III in Season 2, but Fraction IH only in Season 3 . Assessments were made using a Cladosporium spp. bioassay, visible light, short- and long-wave ultraviolet light and vanillin acid test.
T a e bl 8 1 S f e ame la It an d A ssessment 0
Species (Season) Fraction 1I Free Phenolics'
a b c d e
C. 'Desire' � � � � �
Cjaponica 'Elegans - - - - -
C. pitardii var. pitardii - - - - -
C. saluenensis x C. - � � � - reticulata 'Brian' Resistant C. � � � � � c. - - - - - - � � � - C. lutchuensis - - - - - C. - - - - - f F ractlOn 11 an d I I Ph eno lC r C d s Fraction III f a b c d e f � � � � � � � - � � .., � - � - � � .., � - - � � � � � - � � � � .., � � � - � � .., � - - � � .., .., � - .., - � � .., � - - - � � .., � - -
a letters a-frefer to assessment method as descnbed on the preVIOUS page
8.2. 1 .4 Analysis
Zones of inhibition found in the bioassay were considered the most important indication that antifungal phenolic compounds were present in a sample and all other assessment methods were compared against this. Because of likely variability between samples prepared in different seasons and differences occurring during the development of each chromatograph plate, bands present within Rf ± 0. 1 0 of
inhibition zones were considered to be of further interest as possibly containing antifungal phenolic compounds.
A visual companson of developed chromatograph plates with each assessment method was made initially. The various bands and zones of inhibition produced appeared to be similar between resistant and susceptible species. No bands were produced by Fraction II free phenolic samples and testing of this Fraction was discontinued in Season 3 .
Rr values of each species over the four time samples were compared for each assessment method to determine whether bands were constitutively present or increased/decreased over time. Rr values of each species at each time sample for each assessment method were compared to determine whether similar bands were present or absent in resistant and susceptible species. Small imperfections in the production of the silica gel plates led to some variation in Rr values between samples on the same plate (Figure 8. 1 ). Where bands were obviously similar but the Rr value distorted by these imperfections is indicated in the result tables.
2 3 4 5
Figure 8. I
6
Solvent front
Developed chromatograph plate under short-wave ultraviolet light showing the effect of imperfections in the silica gel on bands of the same Kf value. Samples are from C. fraterna. Lane I and 2 0 h; Lanes 3 and 4 24 hpi and Lanes 5 and 6 48 hpi samples. The two 24 hpi samples show considerable distortion in both orientation and Kf value when compared to 0 hpi and 48 hpi samples.
8.2.2 Cell Wall Appositions and Lignification of Existing Cell Walls
8.2.2. 1 Preparation and Storage of Samples
Petals of susceptible and resistant camellia flowers, rhododendron and magnolia flowers (Table 8.2) were collected and processed during the week of 1 0.09.02.
Table 8.2 Resistance Rating of Species and Cultivars of Camellias Used in a Study of Cell Wall Modifications Resistance C. UIR C. R C. R C. R 'Desire' S C. S C. lutchuensis R C. lutchuensis Blush' R C. var. S S C. reticu/ata Cha' S
C. saluenensis x c. reticulata 'Brian' S
C. transnokoensis R
C. UIR
C. R
C. R
stellata non-host resistance
Rhododendron non-host resistance
• S=susceptible, R=resistant, UIR=untested, observations indicate resistant
For each species, there were four treatments: 0 h (uninoculated control), 24 h, 48 h and 72 h after inoculation with ascospores of C. camelliae. Petals were inoculated using the airborne inoculation chamber (Section 6.2.2) then transferred to a humid chamber and incubated at 20°C. At 24 h intervals, five to six petal discs ( 1 2 mm diameter) were cut using a cork borer (or whole petals were used for C. forrestii).
Discs were cut from fresh uninoculated petals (0 h control) immediately after collection. The discs were cleared in 1 50 ml beakers containing 50 ml of 1 M KOH which were autoclaved for 1 5 min at 1 2 1 °C and stored at 4°C until after the camellia season had finished. Four petals of susceptible C. saluenensis x C. reticulata ' Brian' were included in every inoculation to ensure that the C. camelliae ascospores were infective. These petals were transferred to the humid chamber along with the sample petals and incubated until large C. camelliae lesions were evident. Sample petals may have been infected naturally prior to collection.
8.2.2.2 Toludine Blue 0 to Stain Lignin
Prior to staining, petal discs were rinsed thrice in RO water for 5 min each rinse. Discs were not blotted and were handled as little as possible since they were extremely fragile.
After rinsing, two discs from each sample were stained in Toludine Blue 0 (Section 2.5.2) for between 40 min and two hours. Discs were removed from the stain and rinsed once in RO water. Excess stain and water were removed using a tissue laid next to the disc to drain liquid by capillary action. Discs were floated on to microscope slides, a drop of Shears Mounting Fluid added and then a coverslip. Slides were viewed with brightfield microscopy using an Olympus BH-2 microscope.
8 .2.2.3 Analysis
Each disc was scanned at 400x magnification for the presence of ascospores, papillae, lignitubers and signs of cell wall lignification.
8.3 RESULTS
8.3.1 Phenolic Compounds
Control diethyl ether spots run on each silica gel plate did not produce bands on any of the plates.
8.3. 1 . 1 Fraction 11 Samples
Testing of Fraction II free phenolic samples was discontinued after the results of the first two seasons' tests. None of the samples from the two susceptible or two resistant species produced zones of inhibition in the Cladosporium spp. bioassay or bands using the other assessment methods. Occasionally a short smear was present at the spot site, or a very faint band, but as seen in Figure 8.2, the band was barely distinguishable.