Data Analyses

For green shoot inoculation, lesion measurements were adjusted by deducting the mean length of wound and necrotic edge on negative controls (4 mm) from the lesion lengths of each shoot. Since assessment periods for each batch differed, ratios of lesion lengths with respect to the mean lesion lengths shown by the positive control for each batch were calculated and included in the analysis.

For cane inoculation, the experiment ran for six months (spring to summer) and the

environmental conditions differed in each month. Thus, ratios of lesion lengths with respect to positive control for each batch were also calculated and included in the analysis.

For the analysis, the adjusted data of green shoot lesion lengths, the cane lesions lengths, their calculated ratios with respect to the positive control, and corresponding re-isolation distances of canes were tested for homogeneity using Levene‟s test at P≤0.05. When

necessary, log10 transformation was done to improve homogeneity of variance (Gomez & Gomez, 1984). Within each assay, isolate and botryosphaeriaceous species differences with respect to green shoot and cane lesion lengths and ratios, and re-isolation distances, were determined by one-way ANOVA using SPSS 17 Statistics. Means that were significant at

P≤0.05 were separated by pairwise comparisons using least significant differences (LSD) at

the 5% significance level. Lesion means of the positive controls from each batch were compared for each assay to determine variation between batches. Green shoot and cane lesion lengths, ratios relative to the positive controls and re-isolation distances of N. luteum

and N. parvum isolates were separately analysed by one-way ANOVA to determine effects of nursery sources. Means were separated by LSD at P≤0.05. Differences in means between cane lesions and re-isolation distances for each species were determined using t-tests at P≤0.05 level.

Additionally, replication means for isolates of the same species, but only those with eight or more isolates, were graphed on scatter plots using Microsoft Excel 2010. This indicated the potential for linear relationships between cane lesion lengths and re-isolation distances, as well as between green shoot lesion lengths and cane lesion lengths. The extents of the linear relationships between the said variables were determined by coefficient of determination (R2) values.

3.1.3 RESULTS

3.1.3.1 Detached green shoots

Five to seven days after inoculation, green shoots inoculated with all botryosphaeriaceous isolates, except for one isolate each of B. dothidea (D047) and D. seriata (O100), exhibited dark to light brown lesions that developed both upward and downward from the inoculation point (Figure 3.1). Some lesions, including those from positive controls, developed rapidly and encircled the stem leading to wilting leaves and eventually death of shoots within 5 days (Figure 3.1A). Other lesions only progressed on the inoculated side of the stem and the leaves remained normal for up to 7 days (Figure 3.1B and C). Negative control plants from all nine batches were free of lesions beyond the wound necrosis. Mean lesion lengths of the positive control differed significantly (P<0.001; Appendix C.2.1) between batches, with Batch 6 having the longest mean lesion of 135.75 mm while Batch 2 had the shortest mean lesion of 18.75 mm.

Colonies similar to the inoculated botryosphaeriaceous isolates were consistently isolated from the four randomly selected green shoots and positive control shoots, while no

pathogens were isolated from negative control shoots for each batch.

Figure 3.1 Green shoots inoculated with botryosphaeriaecous isolates exhibiting lesions that extended upward and downward (red arrows) from the inoculation point (black arrows).

Lengths of lesions were significantly different for the seven botryosphaeriaceous species and the controls (P<0.001; Table 3.1; Appendix C.2.2). Six of the seven

botryosphaeriaceous species caused mean lesion lengths that were significantly longer (P≤0.05) than for the negative control (0.0 mm), while six out of seven species caused mean

lesion lengths that were significantly shorter (P≤0.05) than for the positive control (mean

83.5 mm). Lesion lengths were greatest for N. parvum (P≤0.05), with a mean of 78 mm that

was significantly (P≤0.05) longer than those of all other species and equally pathogenic to

that of the positive control (N. parvum P069). Neofusicoccum luteum and N. australe were equally pathogenic with mean lesion lengths of 55 and 52 mm respectively, that were

significantly longer than for D. mutila (29 mm) and D. seriata (9 mm). The mean lesion length for D. seriata was not significantly different from that of the negative control. The mean lesion length of the single N. macroclavatum isolate was not significantly different from those of other species except for N. parvum.

The ratios of shoot lesion lengths with respect to mean length of positive control lesions also varied significantly among species (P<0.001; Appendix C.2.2) and generally showed similar species effects to green shoot mean lesions, with N. parvum having the highest mean ratio (0.90) that differed significantly (P≤0.05) from those of all other species except for

N. australe (0.85). The ratio means of B. dothidea (0.30), D. mutila (0.24) and D. seriata

(0.12) were significantly lower than for the other three species. The ratio mean of the single

N. macroclavatum isolate (0.37) was significantly different from that of N. parvum but not those of the other species.

Lesion lengths also varied among isolates of the six botryosphaeriaceous species

(Appendices C.2.4 to C.2.9). Since there was only one isolate for N. macroclavatum, it was excluded from the analysis. Mean lesion lengths varied significantly between isolates of

N. luteum (P<0.001), with means ranging from 3 to 115 mm. For N. parvum, mean lesion lengths also varied significantly (P=0.035) among isolates, with means ranging from 6 to 122 mm while the eight N. australe isolates also differed significantly (P<0.001), with means ranging from 10 to 104 mm. Mean lesion lengths among D. mutila and B. dothidea isolates also differed significantly (P<0.001), with means ranging from 3 to 85 mm and 0 to 104 mm, respectively. For D. seriata, mean lesion lengths of the four isolates also differed significantly (P=0.044), with means ranging from 0 to 26 mm.

The mean ratios of isolate lesion lengths relative to the positive control also varied significantly among isolates within species (P<0.001 to P=0.046) except for D. seriata

Table 3.1 Mean lesion lengths and mean lesion length ratios relative to the positive control that were caused by isolates of botryosphaeriaceous species inoculated onto green shoots.

Species No. of isolates A Mean Lesion length (mm) Range of lesion means (mm) A Ratio with positive control Range of ratio means N. luteum 67 55.4 b 3-115 0.78 b 0.04-2.08 N. parvum 21 77.7 a 6-122 0.90 a 0.42-1.42 N. australe 8 52.2 bc 10-104 0.85 ab 0.14-2.18 D. mutila 8 28.8 de 3-85 0.24 c 0.40-0.64 B. dothidea 6 36.3 cd 0-104 0.30 c 0.00-0.78 D. seriata 4 9.1 ef 0-26 0.12 c 0.00-0.23 N. macroclavatum 1 40.5 bcde 2-58 0.37 bc 0.02-0.53 Positive control 1 83.5 a 19-136 Negative control 1 0.0 f 0 A

Means within a column with different letters are significantly different at P≤0.05 LSD

3.1.3.2 One-year-old rooted canes

External lesions were not observed on inoculated canes, and all inoculated plants showed normal shoot development that did not differ from the negative controls 28 days after

inoculation. However, when the bark was peeled from the trunks, symptoms which appeared as light streaking to dark brown lesions of varying lengths, were observed on all inoculated plants (Figure 3.2), while some negative control plants exhibited light brown streaking just below the inoculation point. Some lesions near the inoculation point had encircled the stem while some lesions only progressed on one side of the stem. Mean lesion lengths of the positive control differed significantly (P=0.002; Appendix C.2.1) between batches, with Batch 3 having the highest mean lesion of 157.5 mm while Batch 7 had the shortest mean lesion of 32.5 mm.

Figure 3.2 Canes inoculated with botryosphaeriaecous isolates exhibiting lesions (red arrow) of different lengths below the inoculation point (black arrow) with normal shoot development (blue arrow).

Lengths of cane lesions were significantly affected (P<0.001) by inoculation treatments as shown in Table 3.2 (Appendices C.2.2). Six of the seven species tested produced lesions that were significantly longer than for the negative control (P≤0.05), and N. parvum was the most pathogenic with the longest mean lesion length of 82 mm, which differed significantly (P≤0.05) from all other species and was equally pathogenic as the positive control

(N. parvum P069). The mean lesion length caused by D. mutila (63 mm) was significantly (P≤0.05) longer than for N. luteum (41 mm) and B. dothidea (18 mm) but did not differ significantly from those caused by N. australe (43 mm)and D. seriata (43 mm). The mean lesion length caused by N. luteum did not differ significantly from those of N. australe and D. seriata but were significantly longer (P≤0.05) than for B. dothidea (18 mm), which produced the shortest mean lesion that did not differ significantly from that of the negative control plants (3 mm). The mean lesion length of the single isolate of N. macroclavatum (52 mm) was significantly shorter than for N. parvum (P≤0.05) but did not differ from those of the

other botryosphaeriaceous species tested.

The ratio of cane lesions with respect to mean length of positive control lesions also varied significantly among species (P<0.001; Appendix C.2.2) and showed similar species effects to cane lesion lengths except that no significant differences were observed between

Table 3.2 Mean lesion lengths and mean lesion length ratios relative to the positive control caused by isolates of botryosphaeriaceous species inoculated onto one-year-old canes.

Species No. of isolates A Mean Lesion length (mm) Range of lesion means (mm) Ratio with positive control A Re-isolation distances mean (mm) Range of re-isolation distances (mm) N. luteum 67 40.9 c 4-125 0.67 b 47.1 c 10-150 N. parvum 21 82.0 a 14-193 1.12 a 94.6 a 30-180 N. australe 8 43.3 bc 8-89 0.67 b 55.0 bc 32-85 D. mutila 8 62.8 b 25-138 0.77 b 59.4 b 33-105 B. dothidea 6 18.2 de 0-34 0.31 c 37.0 c 23-56 D. seriata 4 42.9 bc 30-55 0.53 bc 23.1 cd 18-30 N. macroclavatum 1 52.3 bcd 32-67 0.80 abc 67.5 bc 30-100 Positive control 1 75.4 a 33-158 85.3 a 33-65 Negative control 1 3.1 e 0-8 0.0 d 0 A

Means within a column with different letters are significantly different at P≤0.05 LSD

Analyses of cane lesions caused by isolates of the six botryosphaeriaceous species are summarised in Appendices C.2.4 to C.2.15. Mean lesion lengths on canes varied

significantly among isolates of N. luteum and N. parvum but not among the isolates of other species. For N. luteum isolates, the mean lengths of lesions varied significantly (P<0.001), with means ranging from 4 to 125 mm. For N. parvum, mean lesion lengths also differed significantly (P<0.001) among isolates, with replication means ranging from 14 to 193 mm. Variances of cane lesion means among N. australe isolates were not homogenous

(P=0.321) and transformation did not improve homogeneity, thus, data were excluded from the ANOVA. However, means of lesion lengths among the eight N. australe isolates ranged widely from 8 to 89 mm. Mean lesion lengths among the eight D. mutila isolates did not differ significantly (P=0.141), with means ranging from 25 to 138 mm. Mean lesion lengths also did not differ significantly (P=0.538) among the six B. dothidea isolates, although isolate D047 produced no lesions while D445 caused a mean lesion length of 34 mm. For D. seriata,

mean lesions lengths of the four isolates also did not differ significantly (P=0.800) with means ranging from 30 to 55 mm. There was only one isolate of N. macroclavatum and so it was excluded from the analysis.

The mean ratios also varied significantly among isolates for N. luteum and N. parvum

(P≤0.001) and generally showed similar isolate effects to cane mean lesion lengths. The mean ratios among isolates of the four other species did not differ significantly (Appendices C.2.6 to C.2.9).

Similar to cane lesion lengths, re-isolation distances also differed significantly (P<0.001) between species, with N. parvum having the longest re-isolation distance mean at 95 mm that differed significantly (P≤0.05) from those of the other six species (Table 3.2; Appendix

C.2.1). Mean re-isolation distances of D. mutila (59 mm), N. australe (55 mm), N. luteum (47 mm) and B. dothidea (37 mm) did not differ significantly between species. The mean re- isolation distance for D. seriata (23 mm) was significantly lower than for N. parvum and

D. mutila (P≤0.05) but not for other species. For the positive control plants, the mean re-

isolation distance (85 mm) differed significantly from those of all other species, except for

N. parvum, while no pathogens were re-isolated from negative control plants. The mean re- isolation distance of the single isolate of N. macroclavatum was 68 mm and was significantly shorter than for N. parvum, but did not differ from those of the other botryosphaeriaceous species tested.

Means for re-isolation distances among isolates of the six botryosphaeriaceous species are summarised in Appendices C.2.10 to C.2.15. Significant differences in re-isolation distance means were observed among isolates of two out of six species tested. Re-isolation

distances varied significantly among N. luteum isolates(P<0.001),with means ranging from 10 to 150 mm. For N. parvum isolates, re-isolation distances differed significantly (P<0.001) among isolates, with means ranging from 30 to 180 mm. For N. australe, variances of re- isolation distance means among isolates were not homogenous (P=0.061) and log10 transformation did not improve the homogeneity, thus, data were not analysed by ANOVA. Similar to cane lesions, no significant differences were observed for re-isolation distances among D. mutila (P=0.727), B. dothidea (P=0.280) and D. seriata (P=0.862). However,

B. dothidea isolate B047 which did not produce any cane lesions was re-isolated up to 23 mm from the inoculation point. In contrast to the other five species, the four D. seriata

isolates were only re-isolated from the older parts of the lesions and none were re-isolated from the younger parts or beyond the lesions.