5.4 Results
5.4.2 AM fungal colonisation and sporulation in trap roots
µm mesh) and then separated from foreign particles by centrifugation (2000 rpm for 2 minutes) in a 70% sucrose solution, following the methods of Gerdemann and Nicolson (1963). After staining with trypan blue for 24 h, the spores were mounted on a membrane filter with a 3 mm squared grid and counted as described in Chapter 2.4.
5.3.6 Statistical analysis
Provided that results passed the test for normal distribution (Kolmogorov-Smirnov test; p >
0.05) and homogeneity of variance (Levène test; p > 0.05), data were subjected to a one-way ANOVA. The multiple comparison Tukey-test was used to estimate differences between means. In both tests, p values below 0.05 were interpreted as indicating significant effects. Data which did not show homogeneity of variance was subjected to the Kruskal-Wallis-test (p <
0.05). Statistic calculations were conducted using SPSS software, version 15.0 (SPSS Inc., USA). Results in tables and figures are presented as treatment means ± standard deviation.
Fig. 5.3: Microphotographs of trap roots after 14-days incubation within the substrate of pre-cultivated maize plants inoculated with Glomus mosseae in [viableAM] treatments (experiment 1 and 2). Lateral hyphae growth and differently sized spores found between cortical cells of: a. ’Rmc’ tomato, and b. Tropaeolum trap roots. c.
Intercellular hyphae growth longitudinal to the trap root cortex of T. majus, as generally found for all studied trap root genotypes. d. Trap roots originated from ‘Pak Choi’ with diameters smaller than 150 µm, colonised extra-radically with spores and hyphae. e. A coarse trap root of C. gayana colonised intra-extra-radically by AM fungal spores.
Stelar cylinders were frequently colonised by AM fungal spores, shown in a dissected Tropaeolum sample (f) and in an intact C. gayana sample (g). h. Top view of a nylon mesh layer, excised from a stained mesh control. Coarse and finely branched hyphae were observed to cross the mesh surface but no spores were found. All trap roots were free from any fungal colonisation before being used for incubation. Bars indicate 100 µm.
a
d
f g h
e
b c
In [viableAM] treatments, hyphae growth of G. mosseae (diameter of hyphae 3 to 15 µm) was observed on the surface and along the trap root main axis (Fig. 5.3 a, see arrows), as well as between cortical cells (Fig. 5.3 c). Hyphae were spread all through the trap root tissue and showed branching, and occasionally, inter-connection by h-bridges. G. mosseae developed thick-walled, oval or globose shaped spores (diameter up to 150 µm) within the cortex (Fig. 5.3 a, b and e). To a lesser extent globose shaped spores were also observed outside of the trap root tissue (Fig 5.3 d) and within the stelar cylinder (Fig 5.3 f and g). No arbuscules were found in trap roots.
0 20 40 60 80 100
76R tomato rmc tomato Tropaeolum Pak Choi
0 20 40 60 80 100
76R tomato rmc tomato Tropaeolum Pak Choi
0 5 10 15
76R tomato rmc tomato Tropaeolum Pak Choi
Fig. 5.4: Percentage of AM fungal colonised trap root length and ratio of percentage hyphal-to-spore colonisation (Experiment 1). Trap roots from different plant genotypes (named on x-axis) were inserted for a 14-day period into a substrate containing a pre-cultivated maize plant inoculated with the AM fungus Glomus mosseae. a.
Percentage of root length with AM fungal hyphae colonisation. b. Percentage of root length colonised with intra-radical spores. Here, spores outside of the root cortex were not counted. c. Ratio of percentage hyphal-to-spore colonised root length. Bars represent means ± SD. Different letters indicate significant different means (multiple comparison Tukey-test, p < 0.05; n = 4).
AMF hyphal colonisation (% of trap root length) a
b
AMF intra-radical spore colonisation (% of trap root length)
Ratio of percentage hyphae-to-spore colonised trap root length
a
a a
b
‘WT’ ‘rmc’ T. majus ‘Pak Choi’
Tomato c
a a a a
b b b
a
Trap roots originated from wild-type tomato (host), Tropaeolum (host) and rmc mutant tomato (non host), did not differ in colonisation rates and averaged between 55 - 80% for hyphal colonisation and between 20 - 30% for intra-radical spore colonisation (Fig. 5.4 a and b). For
‘Pak Choi’ (non host), the trap root length colonised with spores was up to 12% which was significantly lower (Fig. 5.4 b) than for the other trap root genotypes. In addition to this, the pattern of colonisation in ‘Pak Choi’ was different than in the other genotypes. The cortex of
‘Pak Choi’ trap roots were colonised with many spores and hyphae (see Fig 5.3 d), while intra-radically, hyphae dominated and relatively few spores were present. This pattern was also reflected in the high ratio of hyphae-to-spores in ‘Pak Choi’ roots (Fig. 5.4 c).
The spore density per unit length of trap roots originated from ‘Pak Choi’ was lower than for the other trap root genotypes (Fig. 5.5 a). A similar result was also observed for spore density per unit trap root DW (Fig. 5.5 b).
0 5 10 15 20
76R tomato rmc tomato Tropaeolum Pak Choi
0 20 40 60 80 100
76R tomato rmc tomato Tropaeolum Pak Choi
Fig. 5.5: Intra-radical spore densities per unit trap root (Experiment 1), estimated as a. per cm trap root length and b. per mg trap root DW. Bars represent means ± SD. Different letters indicate significant different means (multiple comparison Tukey-test, p < 0.05; n = 4).
In [viableAM], the trap roots of C. gayana had a specific root length of 221 ± 155 m g-1 and an average total length of 11 ± 0.3 m per compartment. The total AM fungal colonised length of trap roots averaged 39 ± 4%, with 13 ± 2% intra-radical spores.
a
AMF intra-radical spore density (no. cm-1 trap root length)
b b
b
a
b b b
a
‘WT’ ‘rmc’ T. majus ‘Pak Choi’
tomato b
AMF intra-radical spore density (no. mg-1 trap root DW)
Within distinct root diameter size classes, the total trap root length in the compartment, the trap root length colonised with AM intra-radical spores, and the trap root length not colonised with AM intra-radical spores, were estimated for each genotype used in experiments 1 and 2. Since both wild-type and rmc tomato trap roots were similar in their features and were colonised in a similar way with AM fungal structures, data for rmc roots are not shown any further. Roots were separated into three diameter size classes, defined as <150 µm (fine), 150-300 µm (intermediate) and >300 µm (coarse) (Fig 5.6 a-d). Tomato roots predominantly consisted of diameters larger than 150 µm (Fig 5.6 a) with a very low proportion of roots being thinner than 150 µm. This trend could also be observed in T. majus trap roots (Fig 5.6 b) which were completely lacking the finest root class. In contrast to this, trap roots originated from Pak Choi and C. gayana showed a relatively high proportion of thin diameter classes (37% and 30% of total length in compartment; Fig. 5.6 c and d). C. gayana trap roots exhibited a relatively homogenous distribution pattern of all size classes. The percentage of trap root length colonised with intra-radical spores (Fig 5.6 e-h) was low in fine trap roots and markedly increased with increasing diameter size. This trend was observable in all studied genotypes, except for T.
majus trap roots which lacked of the thinnest diameter size class (Fig. 5.6 f), but was most pronounced in Pak Choi and C. Gayana (Fig. 5.6 g and h). Both these genotypes, who exhibited root size classes in approximately the same dimensions, showed that up to four times more coarse trap root length was colonised with intra-radical spores than for the fine class.
0 2 4 6
ø (µm) <=140 150-300 >=310 mean class partitio n o f to tal ro o t length (m/ trap patch)
0 20 40 60 80 100
ø (µm) <=140 150-300 >=310 mean % partitio n spo re colo nized within diam.
class
0 2 4 6
ø (µm) <=140 150-300 >=310 mean class partitio n o f to tal ro o t length (m/ trap patch)
0 20 40 60 80 100
ø (µm) <=140 150-300 >=310 mean % partitio n spo re co lo nized within diam. class
0 2 4 6
ø (µm) <=140 150-300 >=310 mean class partitio n o f to tal ro o t length (m/
trap patch)
0 20 40 60 80 100
ø (µm) <=140 150-300 >=310 mean % partitio n spo re co lo nized
within diam. class
0 2 4 6
ø (µm) <=140 150-300 >=310 class partitio n of to tal ro o t length (m/ trap patch)
0 20 40 60 80 100
ø (µm) <=140 150-300 >=310 mean % partitio n spo re co lo nized within diam.
class
Fig. 5.6: Total trap root length (m per compartment) (figures on the left) and percentage of trap root length colonised with AM fungal intra-radical spores (figures on the right). Values were estimated for each root diameter size (see x-axis). Trap roots were excised from the plant species indicated on top of the respective diagram. Bars represent means ± SD. Different letters (figures on the right) indicate significantly different means (multiple comparison Tukey-test, p < 0.05; n = 4). Data were square root transformed prior to statistical analysis.
< 150 µm 150-300 µm > 300 µm
WT tomato
T. majus
< 150 µm 150-300 µm > 300 µm a
c b
d
e
f
g
h
a
b b
a
b
c
a a
a
b b
Exp. 1 Exp. 2
Total trap root length (m per compartment)
Pak Choi
C. gayana
Trap root length colonised with AM fungal intra-radical spores (%)
WT tomato
T. majus
Total trap root length (m per compartment) Trap root length colonised with AM fungal intra-radical spores (%)
Pak Choi
C. gayana
Experiment 3
5.4.3 Nurse plant root AM fungal colonisation and spore density in pot substrate