5.5.5.1 Materials and methods
For this experiment four treatments (3–6) were carried out in the four waterbath chambers during four consecutive runs over a period of 16 days from 22 November. Petal from treatments 1 and 2 were incubated in 98 and 85% RH saturated salt chambers at 20oC, respectively, and were repeated during each waterbath treatment. Each treatment had set points and actual conditions (measured during the first run of each treatment) as shown in Table 5.2.
Pistillate flowers were collected from six vines in orchard 20 on 22 and 25 November 1997 and from six vines in orchard 21 on 30 November and 5 December 1997 (Appendix 1). The whole experiment was repeated once with flowers sampled from six vines in orchard 6 on 9, 15, 19 and 22 December. Six flowers were collected from each vine on each sampling date. Two petals were taken at random from the flowers of each vine for each of five batches of inoculation. S. sclerotiorum ascospores from 16 grids of membrane filter disc were used to inoculate each batch. The 12 petals from each inoculation batch were placed into micro-tube holders and two were randomly assigned to one of six incubation chambers i.e. the two saturated salt chambers (treatments 1 and 2) and the four chambers within the waterbath. Petals were positioned within chambers as shown in Figures 5.12 and 4.5.
Three un-inoculated control petals were taken at random from the flowers immediately after each of the five batches of inoculation were completed. A sixth set of three control petals was taken after the fifth inoculation batch. These were placed directly into micro-tube holders and randomly assigned to one of the six chambers. All petals were removed from the chambers after 70–72 h incubation and assessed for percentage area of lesions before surface-sterilisation. Inoculated petals from each treatment were combined into pairs (Figure 5.12) and macerated together in either 2, 3 or 4 ml PBS depending on the extent of soft rot within each treatment. Un-inoculated control petals from within each chamber were macerated together in 3 ml PBS. Two 50 Pl aliquots from each sample were spread onto a Petri dish of JK selective medium and cfu were determined (section 4.3.4).
Chapter 5 Factors affecting colonisation of detached petals by ascospores 117
Table 5.2 Set points, mean, standard deviation and range of temperature and relative humidity conditions for treatments 1–6 during the first run of Experiment 5-10.
Treat- ment
Temperature x Relative humidity x Set point (oC) Actual conditions (oC) Set point (%) Actual conditions (%) 1 20 19.3 r 0.4 1 (18.7–19.8)2 98 98 r 0.5 1 (97–99)2 2 20 19.6 r 0.4 (18.9–19.8) 85 87 r 0.5 (85–89) 3 20 20.0 r 0.09 (19.9–20.3) 97 95 r 2.1 (91–99) 4 20 19.8 r 0.09 (19.6–19.9) 85 83 r 1.6 (81–87) 5 20 19.7 r 0.14 (19.4–20.1) 70100 83 r 7.4 (69–96) 6 3 1020 14.8 r 2.7 (10.2–19.2) 70100 88.5 r 8.8 (73–100) 1 Standard deviation. 2 Range in parentheses. 3
See Appendix 4 for graphs of temperature and RH.
NB. for treatment 1 and 2, n = 8 and for treatments 36, n = 864 (5 minute readings). The mean temperature and RH for the period October–December 1996 at the Te Puke Research Orchard weather station was calculated for each hour of the day. This diurnal pattern of temperature and RH was used to model the conditions within the waterbath chambers during treatments 5 and 6 (Appendix 4), by programming changes to the set points of waterbath 1 and 2 (Appendix 7).
Subsequently, the diurnal pattern of temperature and RH for the period 1 November to 31 January 1996-99 was calculated from the available hourly data at the Te Puke Research Orchard weather station. The data was separated into a November–December period, coinciding with kiwifruit flowering (n = 130 days) and a January–February period, coinciding with early fruit development (n = 137 days). Each period of data was split into days with t5 mm rainfall day-1 (22 days for November–December and 18 days for January–February) and days with <5 mm rainfall day-1. The threshold of 5 mm was arbitrary, but approximates the rainfall required to thoroughly wet a vine canopy.
Absolute humidity was calculated for each hour of the day for each of the four data sets using Psychrometric Function software (PKSFX ver. 2.5 demo, PKWARE Inc.). The dry bulb temperature and absolute humidity for each of the above data sets was then
Chapter 5 Factors affecting colonisation of detached petals by ascospores 118
used to plot treatment and field environmental conditions on a simplified psychrometric chart (section 5.5.7, Figure 5.20).
Following angular transformation the percentage area of lesions and log10
transformation of cfu/petal (+1), data was analysed by REML (Residual maximum likelihood) (Patterson & Thompson, 1971) using Genstat. Treatment differences were assessed by likelihood ratio tests (Welham & Thompson, 1992).
5.5.5.2 Results
The mean percentage area of lesions was significantly greater (P<0.01) on petals in treatment 1 (98% RH ‘static’) than in treatment 3 (97% RH waterbath) (Figure 5.17). The mean percentage area of lesions on petals in treatments 2 and 4 (85% RH) and from treatments with diurnally fluctuating conditions was <5% and significantly less (P<0.001) than on petals in treatments 1 and 3. The number of S. sclerotiorum cfu/petal recovered from petals in treatment 1 (390 cfu/petal) was also significantly greater (P<0.05) than from petals in treatment 3 (80 cfu/petal) (Figure 5.18). All other treatments had <5 of cfu/petal S. sclerotiorum and there were no cfu from un-inoculated (control) petals.
There was no significant difference (P>0.05) in the number of B. cinerea cfu/petal recovered from inoculated and un-inoculated petals. There were significantly higher (P<0.05) numbers of B. cinerea cfu/petal in treatment 1 and 3 (97% RH) than the other treatments.
Chapter 5 Factors affecting colonisation of detached petals by ascospores 119
Figure 5.17 Mean percentage area of lesions on petals inoculated with S.
sclerotiorum ascospores and incubated at 20oC for 72 h at 98% RH and 85% RH in
saturated salt chambers (static), 97% RH, 85% RH, fluctuating RH (70–100%), and fluctuating RH (70–100%) and temperature (10–20oC) in waterbath chambers. Data is back transformed after angular transformation.
Figure 5.18 Mean cfu/petal of S. sclerotiorum and B. cinerea (log scale) on petals inoculated with S. sclerotiorum ascospores and incubated at 20oC for 72 h at 98% RH and 85% RH in salt chambers (static), 97% RH, 85% RH, fluctuating RH (70– 100%), and fluctuating RH (70–100%) and temperature (10–20oC) in waterbath chambers.
Incubationconditions
97% RH (static)85% RH (static)97% Waterbath85% WaterbathFluctuating RH
Fluctuating RH + Temp
Mean percent area of lesions
0 10 20 30 40 50 60 =Maximumsed Incubationconditions
98% RH (static)85% RH (static)97% Waterbath85% WaterbathFluctuating RH
Fluctuating RH + Temp
Mean cfu/petal
0 10 100 1000 S.sclerotiorum B.cinerea =MaximumsedChapter 5 Factors affecting colonisation of detached petals by ascospores 120