SUPPLEMENTAL INFORMATION FOR:
Plant water stress effects on stylet probing behaviors of Homalodisca vitripennis (Germar)
(Hemiptera: Cicadellidae) associated with acquisition and inoculation of the bacterium
Xylella fastidiosa
RODRIGO KRUGNER AND ELAINE A. BACKUS
United States Department of Agriculture-Agricultural Research Service,
San Joaquin Valley Agricultural Sciences Center, Parlier, CA
Results
Overnight recordings: Group 1 and Group 2 waveforms during nighttime acclimation
period. After transfer from the rearing cages onto test plants for the acclimation period, most
insects remained motionless on citrus or almond plants for an average (± SEM) of 1.36 ± 0.72 or
2.56 ± 0.94 h, respectively, until they probed for the first time. In general, the first stylet probing
behaviors observed were represented by waveforms P and B2, which correspond to formation of
salivary sheath and sheath branching that occur during the search for a xylem cell, respectively.
The mean durations of both waveforms P and B2 per insect (WDI) were significantly longer on
citrus than on almond plants, whereas only the numbers of B2 waveform events per insect
(NWEI) were significantly affected by plant species (Supp. Table S1). Yet, the numbers of
events and durations (both per insect) of waveform P were significantly affected by the irrigation
treatment. Specifically, an average insect performed significantly more and longer pathway
activities in water-stressed than fully irrigated plants (Supp. Table S1). However, plant water
status did not significantly affect the same response variables for the sheath branching behavior
(waveform B2) (Supp. Table S1). Interaction effects were not significant for the above WDI and
NWEI values. The latter result is in contrast to mean waveform P durations per event (WDE),
wherein interaction effects, but not main effects, were significant. Thus, the longest events of P
were performed on water-stressed citrus, the shortest on water-stressed almond, and intermediate
P event durations were performed on fully irrigated plants of both species (Supp. Table S1).
1
The numbers of events and durations per insect for sharpshooter resting behaviors with
the stylets inserted in the plant but motionless (i.e., waveform R, stylets outside xylem;
waveform G, stylets inside xylem) were not significantly affected by irrigation treatment.
However, the duration per insect of waveform G was significantly longer on citrus than on
almond plants (Supp. Table S2). In contrast, events of waveform U (i.e., unknown behavior)
were observed significantly more often and lasted longer (once totaled and averaged per insect
for WDI) in almond than in citrus plants. Irrigation treatment did not significantly affect the
duration of waveform U per insect, but it did significantly affect the number of U events per
insect; a larger number of events of this unknown behavior were performed on water-stressed
than on fully irrigated plants (Supp. Table S2).
Mean durations per events (WDE) for waveform R were significantly different for main
irrigation effects and for interactions; thus, R events were longest on water-stressed citrus, with
all other treatment combinations not significantly different. Results for G events were different
from R; both plant effects and plant × irrigation interaction effects were significant, revealing
that the longest G events were those recorded on fully irrigated citrus. Events of unknown (U)
waveform were significantly shorter on citrus compared with almond plants, but not different for
irrigation or interaction effects (Supp. Table S2).
Overnight recordings: Group 3 waveforms during nighttime acclimation period.
Irrigation status did not significantly affect the numbers of events and durations (both per insect)
of XN, XC and C2 (Supp. Table S3). However, the numbers of events and durations (per insect)
of waveform XN were significantly lower on citrus than almond plants. Also, while events of
waveform XC were observed significantly less often on citrus than on almond plants, there was
no significant plant species effect on the durations of waveform XC per insect. Neither NWEI
nor WDI for sustained xylem sap ingestion (C2) were significantly affected by plant species or
irrigation treatment during the overnight acclimation period (Supp. Table S3).
Mean event durations (WDE) for all three X wave components were significantly
different for plant species main effects, but not for irrigation or plant × irrigation interactions.
Thus, events of both XN and XC were longer on citrus compared with almond, but events of C2
(sustained xylem fluid ingestion) were longer (accepting a P value of 0.056 as significant).
However, irrigation status did not affect per-event durations of X waves or sustained ingestion
(Supp. Table S3).
2
Daytime recordings: Group 1 and Group 2 waveforms during daytime conditions. In
general, behaviors performed during the search for a xylem cell (waveforms P and B2) were not
affected by plant water stress. On a per-insect basis, there were no differences between fully
irrigated and water-stressed plants in both numbers of events and durations of these pathway
behaviors (Supp. Table S4). However, there was a plant species effect in the P duration per
insect, being longer when performed on citrus than almond plants (Supp. Table S4). There also
was no difference in the B2 duration per insect, or in the number of events of both waveforms P
and B2 between plant species. Nonetheless, when the duration of individual waveform events
(WDE) were analyzed, both plant species and irrigation treatment affected the P event duration.
P events were longer on citrus than almond plants; shorter on water-stressed than fully irrigated
almond plants, and longer on water-stressed than fully irrigated citrus plants (Supp. Table S4).
Similar to the behaviors performed during the search for a xylem cell, sharpshooter
resting behaviors with stylets inserted in the plant (waveforms R and G) were generally not
affected by irrigation treatment or plant species (Supp. Table S4). The exceptions were for the
numbers of events of waveform R per insect, which were larger in water-stressed than in fully
irrigated plants, and the durations of waveform G per insect, which were shorter in almond than
on citrus plants. Host plant effect for waveform U (unknown biological meaning), for the
waveform durations per insect, the number of events per insect, and the durations of waveform
events, were all shorter on citrus than on almond plants (Supp. Table S4). Irrigation treatment
did not affect the overall durations or numbers of events of U per insect, but it did affect the
durations of individual waveform events; U events were shorter on fully irrigated than on waterstressed plants (Supp. Table S4).
Discussion
Overnight recordings: Group 1 and Group 2 waveforms during nighttime acclimation
period. During the overnight acclimation period, plant species had the greatest effect on stylet
probing behaviors. After being placed on test plants, insects remained motionless for about 2 h
before initiating the searching behaviors. Once stylet probing commenced, the average insect
made longer, more frequent events of pathway/searching behavior (waveforms P and B2) on
citrus than on almond plants; when combined, these changes caused more overall
pathway/searching durations per insect, especially on water-stressed citrus. In addition, there
3
were shorter and less frequent events of the unknown behavior (U) on citrus than on almond.
These unknown behaviors were performed throughout the 10-h period.
Overnight recordings: Group 3 waveforms during nighttime acclimation period. Once
an average insect’s stylets reached the xylem, salivation/egestion (XN) and trial ingestion (XC)
behaviors in xylem cells were strongly affected by host plant. All salivation/egestion events, of
the same per-event duration, were performed more often on almond than on citrus; this resulted
in longer durations of the xylem-testing behaviors, per insect. Similarly, fewer trial ingestion
events (of the same per-event duration) were performed on citrus than almond; however, there
was not enough difference to result in a shorter duration, per insect. Neither plant species nor
irrigation treatment significantly affected any measure of sustained xylem fluid ingestion. The
exception was for the WDE, which was longer in almond than citrus plants. However, while
insects performed sustained ingestion of xylem fluid (C2) from both almond and citrus plants,
they did so about four and ten times less than during the daytime ingestions, respectively. Stylet
resting (either in xylem [G] or more shallowly in other tissues of the plant [R]) often followed an
event of sustained egestion; however, stylet resting was performed about the same, regardless of
treatment. Most stylet resting also occurred very late in the first 10-h period, primarily between
0200 and 0600 h, before dawn and lights-on regime. The above behavioral findings on citrus
(compared with almond) match that performed by sharpshooters on a less-preferred host plant, in
which searching and acceptance behaviors require more time and energy than on a highly
preferred plant. An insect’s search ultimately ends in sustained xylem fluid ingestion.
One explanation for the significant plant species effect (in the first 10 h) on pathway/
salivary sheath/ branching behaviors (indicating higher degree of difficulty in searching for a
xylem cell) is that it is more difficult for naïve insects to locate xylem cells in citrus than almond
plants. Our test insects had never experienced feeding on almond or citrus plants before EPG
recording. Physical impediments such as lignification of cell walls and location/depth of xylem
cells could influence the amount of time needed for searching, being more difficult for naïve
insects on citrus than on almond plants. Another explanation for the lack of a significant
irrigation treatment effect during the nighttime recordings is that insects were well fed when
placed on the plants, not having been subjected to a pre-test starvation period. Short nighttime
xylem fluid ingestion may be more important to prevent dehydration and/or starvation than to
provide the energy and nutrients needed for maintenance and reproduction.
4
Supplemental Material
Table S1. Mean (±SEM) numbers of events and durations of P and B2 waveform variables calculated from electrical penetration graph
recordings of Homalodisca vitripennis feeding behaviors during the first 10 h (nighttime) period on fully irrigated and water-stressed
almond and citrus plants
Plant
Irrigation
Waveform duration (sec)
Number of waveform
Waveform duration (sec)
treatment
per insect (WDI)
events per insect (NWEI)
per event (WDE)
P - pathway (excluding B2)
Water-stressed
561.20 ± 79.91 Aa 7a
17.57 ± 3.44
Aa 7a
31.94 ± 3.16 a
123b
Fully irrigated
376.46 ± 131.99 Ab 8
9.50 ± 3.12
Ab 8
39.63 ± 3.81 bc
76
9
23.22 ± 3.46
Aa 9
53.73 ± 4.82 c
209
679.00 ± 162.04 Bb 8
16.13 ± 3.42
Ab 8
42.11 ± 3.80 ab 129
Almond
Water-stressed
1247.73 ± 233.02 Ba
Citrus
Fully irrigated
F
df
P
F
df
P
F
df
P
Plant species effect
8.34
28 0.007
3.71 28 0.064
1.07 533 0.302
Irrigation treatment effect
8.15
28 0.008
6.62 28 0.015
0.43 533 0.511
Plant × Irrigation treatment
10.78 533 0.001
B2 - sheath branching
5
Water-stressed
93.37 ± 18.74 Aa 7
13.29 ± 3.28
Aa 7
7.03 ± 0.31 Aa 93
Fully irrigated
59.61 ± 20.43 Aa 7
7.43 ± 2.83
Aa 7
8.02 ± 0.50 Aa 52
Almond
Water-stressed
108.78 ± 18.07 Ba
9
16.00 ± 2.59
Ba 9
6.80 ± 0.29 Aa 144
Fully irrigated
114.95 ± 22.85 Ba
7
13.86 ± 2.74
Ba 7
8.30 ± 0.90 Aa 97
Citrus
F
df
P
F
df
P
F
df
P
Plant species effect
5.54
26 0.026
4.11 26 0.053
2.75 382 0.098
Irrigation treatment effect
1.64
26 0.211
2.94 26 0.098
2.59 382 0.108
a
Number of insects that performed the feeding behavior per irrigation treatment.
b
Number of waveforms events performed per irrigation treatment.
Different lower case letters in columns indicate significant differences between irrigation treatments (α = 0.05).
Different upper case letters in columns indicate significant differences between plant species (α = 0.05).
6
Table S2. Mean (±SEM) numbers of events and durations of R, G and U waveform variables calculated from electrical penetration
graph recordings of Homalodisca vitripennis feeding behaviors during the first 10 h (nighttime) period on fully irrigated and waterstressed almond and citrus plants
Plant
Irrigation
Waveform duration (sec)
Number of waveform
Waveform duration (sec)
treatment
per insect (WDI)
events per insect (NWEI)
per event (WDE)
R - stylets resting outside xylem
Water-stressed
5920.67 ± 2168.77
Aa 6a
8.17 ± 2.96
Aa 6a
724.98 ±
70.48 a
49b
Fully irrigated
6984.81 ± 3557.05
Aa 6
8.67 ± 2.69
Aa 6
805.94 ± 112.55 a
52
Water-stressed
11758.83 ± 3748.22
Aa 7
9.14 ± 2.47
Aa 7
1286.12 ± 213.58 b
64
Aa 6
3.33 ± 0.71
Aa 6
Almond
Citrus
Fully irrigated
1848.93 ±
350.06
P
F
df
78.95 a
F
df
P
F
Plant species effect
0.01
21
0.927
0.73
21 0.401
0.11 181 0.735
Irrigation treatment effect
1.97
21
0.174
1.19
21 0.288
6.79 181 0.099
Plant × Irrigation treatment
df
554.68 ±
P
7.46 181 0.007
G - stylets resting inside xylem
7
20
Water-stressed
3011.87 ± 1159.65
Aa 7
5.86 ± 1.55
Aa 7
514.22 ±
99.86 a
41
Fully irrigated
2526.74 ±
898.58
Aa 7
6.00 ± 1.30
Aa 8
421.12 ±
80.28 a
48
Water-stressed
9579.30 ± 2520.25
Ba 9
9.67 ± 2.06
Aa 9
990.96 ± 267.41 a
87
Fully irrigated
16528.48 ± 5343.88
Ba 7
8.29 ± 1.92
Aa 7
1994.82 ± 364.23 b
58
Almond
Citrus
F
df
P
F
df
P
F
Plant species effect
18.79
27
0.0002
2.74
27 0.109
7.82 230 0.005
Irrigation treatment effect
1.10
27
0.303
0.05
27 0.817
2.22 230 0.137
Plant × Irrigation treatment
df
P
7.96 230 0.005
U - unknown behavior
Water-stressed
9172.36 ± 2659.14
Aa 6
17.67 ± 3.00
Aa 6
519.19 ±
93.95 Aa 106
Fully irrigated
10953.27 ± 2567.35
Aa 8
10.75 ± 2.99
Ab 8
1018.91 ± 200.27 Aa 86
Water-stressed
3574.56 ± 1946.97
Ba 8
7.88 ± 2.25
Ba 8
453.91 ± 121.15 Ba 63
Fully irrigated
2365.94 ± 1915.31
Ba 7
3.14 ± 0.91
Bb 7
752.80 ± 413.15 Ba 22
Almond
Citrus
Plant species effect
F
df
P
20.62
25
0.0001
F
df
P
14.58 25 0.0008
8
F
df
P
8.36 273 0.004
Irrigation treatment effect
0.25
25
0.620
6.72
25
0.015
a
Number of insects that performed the feeding behavior per irrigation treatment.
b
Number of waveforms events performed per irrigation treatment.
0.93
273 0.335
Different lower case letters in columns indicate significant differences between irrigation treatments (α = 0.05).
Different upper case letters in columns indicate significant differences between plant species (α = 0.05).
9
Table S3. Mean (±SEM) numbers of events and durations of XN, XC and C2 waveform variables calculated from electrical
penetration graph recordings of Homalodisca vitripennis feeding behaviors during the first 10 h (nighttime) period on fully irrigated
and water-stressed almond and citrus plants
Plant
Irrigation
Waveform duration (sec)
treatment
per insect (WDI)
Number of waveform
Waveform duration (sec)
events per insect
per event (WDE)
(NWEI)
XN - xylem contact
Water-stressed
273.15 ± 46.30
Aa 7a
15.43 ± 2.89
Aa 7a
17.70 ± 1.24
Aa 108b
Fully irrigated
189.50 ± 23.23
Aa 8
10.00 ± 1.30
Aa 8
18.95 ± 1.55
Aa 80
Water-stressed
179.70 ± 31.87
Ba 9
7.22 ± 1.26
Ba 9
24.88 ± 2.38
Ba 65
Fully irrigated
136.50 ± 39.17
Ba 8
6.75 ± 2.09
Ba 8
20.22 ± 1.50
Ba 54
Almond
Citrus
F
df
P
F
df
P
F
df
P
Plant species effect
6.01 28
0.020
10.29 28 0.003
9.55
303 0.002
Irrigation treatment effect
3.51 28
0.071
2.08
0.22
303 0.638
28 0.159
XC - trial ingestion
Almond
Water-stressed
934.45 ± 209.38 Aa 7
14.57 ± 2.40
10
Aa 7
64.13 ±
6.13
Aa 102
Fully irrigated
458.92 ± 61.08 Aa 8
9.50 ± 0.96
Aa 8
48.31 ±
4.65
Aa 76
Water-stressed
534.39 ± 120.97 Aa 8
7.00 ± 1.16
Ba 8
76.34 ±
7.52
Ba 56
Fully irrigated
581.40 ± 148.27 Aa 8
6.75 ± 1.77
Ba 8
86.13 ± 12.83
Ba 54
Citrus
F
df
P
F
df
P
F
df
P
Plant species effect
1.22 27
0.278
10.30 27 0.003
16.39 284 < 0.0001
Irrigation treatment effect
1.50 27
0.231
2.11
0.73
27 0.157
284
0.394
C2 - sustained ingestion
Water-stressed
4002.64 ± 1773.76 Aa 7
3.43 ± 0.72
Aa 7
1167.44 ± 321.99 Aa 24
Fully irrigated
4061.02 ± 2185.56 Aa 4
3.00 ± 1.41
Aa 4
1353.67 ± 376.72 Aa 12
Water-stressed
1859.65 ± 743.82 Aa 6
2.50 ± 0.62
Aa 6
743.86 ± 152.80 Ba 15
Fully irrigated
1673.40 ± 548.36 Aa 4
3.00 ± 0.91
Aa 4
557.80 ±
Almond
Citrus
F
df
P
F
df
P
77.68 Ba 12
F
df
P
Plant species effect
0.84 17
0.372
0.15
17 0.702
3.80
59
0.056
Irrigation treatment effect
0.01 17
0.904
0.01
17 0.925
0.12
59
0.727
a
Number of insects that performed the feeding behavior per irrigation treatment.
b
Number of waveforms events performed per irrigation treatment.
11
Different lower case letters in columns indicate significant differences between irrigation treatments (α = 0.05).
Different upper case letters in columns indicate significant differences between plant species (α = 0.05).
12
Table S4. Mean (±SEM) numbers of events and durations of P, B2, R, G and U waveform variables calculated from electrical
penetration graph recordings of Homalodisca vitripennis feeding behaviors during the second 10 h (daytime) period on fully irrigated
and water-stressed almond and citrus plants
Plant
Irrigation treatment
Waveform duration (sec) per
Number of waveform
events per insect
insect (WDI)
Waveform duration (sec) per
event (WDE)
(NWEI)
P - pathway (excluding B2)
Almond
Citrus
Water-stressed
832.99 ± 285.16
Aa 6a
22.33 ±
7.66 Aa 6a
37.30
± 3.27
Aa 134b
Fully irrigated
1136.77 ± 317.68
Aa 8
27.13 ±
5.71 Aa 8
41.91
± 7.79
Ab
217
Water-stressed
1375.16 ± 272.83
Ba 9
28.56 ±
5.75 Aa 9
48.16
± 3.47
Ba
257
Fully irrigated
1953.77 ± 745.99
Ba 9
44.56 ± 12.96 Aa 9
43.85
± 2.69
Bb
401
F
df
P
F
df
P
F
df
P
Plant species effect
4.50
28 0.043
2.23 28 0.146
3.93 1005
0.048
Irrigation treatment effect
1.18
28 0.286
1.59 28 0.218
7.37 1005
0.007
B2 - sheath branching
Almond
Citrus
Water-stressed
171.20 ± 40.34
Aa 4
21.75 ±
5.86 Aa 4
7.87
± 0.39
Aa
140
Fully irrigated
127.58 ± 29.60
Aa 8
17.50 ±
4.61 Aa 8
7.29
± 0.35
Aa
56
Water-stressed
124.75 ± 35.74
Aa 9
19.00 ±
5.04 Aa 9
6.57
± 0.28
Ba
171
Fully irrigated
229.97 ± 88.97
Aa 9
31.67 ± 11.44 Aa 9
7.26
± 0.30
Ba
285
F
Plant species effect
0.05
df
P
F
26 0.821
df
P
0.26 26 0.615
13
F
df
P
8.25
679
0.004
Irrigation treatment effect
0.01
26 0.908
0.08 26 0.779
1.06
679
0.304
R - stylet resting outside xylem
Almond
Citrus
Water-stressed
6990.79 ± 3729.49 Aa 4
7.75 ± 3.94 Aa 4
902.04
±
91.45
Aa
31
Fully irrigated
3796.70 ± 1650.21 Aa 6
5.33 ± 1.54 Ab 6
711.88
± 101.99
Aa
32
Water-stressed
8141.26 ± 1687.87 Aa 7
10.00 ± 2.33 Aa 7
814.13
± 108.65
Aa
70
Fully irrigated
3293.91 ± 1428.87 Aa 6
2.33 ± 0.61 Ab 6
1411.67
± 665.34
Aa
14
F
df
P
F
df
P
F
df
P
Plant species effect
0.34
19 0.560
0.02 19 0.895
0.71
143
0.402
Irrigation treatment effect
1.97
19 0.176
4.22 19 0.054
0.90
143
0.344
Water-stressed
2881.23 ± 1211.95 Aa 6
6.00 ± 2.56
Aa 6
480.20
±
80.69
Aa
36
Fully irrigated
3950.43 ± 1861.73 Aa 8
6.63 ± 1.82
Aa 8
596.29
± 118.55
Aa
53
Water-stressed
7020.84 ± 1961.78 Ba 9
9.67 ± 2.51
Aa 9
726.29
± 115.51
Aa
87
Fully irrigated
9366.97 ± 2246.08 Ba 9
8.67 ± 2.17
Aa 9
1080.80
± 166.17
Aa
78
G - stylet resting in xylem
Almond
Citrus
F
df
P
F
df
P
F
df
P
Plant species effect
9.04
28 0.005
1.90 28 0.179
0.15
250
0.703
Irrigation treatment effect
1.77
28 0.194
0.00 28 0.954
0.05
250
0.815
U - unknown behavior
Almond
Citrus
Water-stressed
13671.95 ± 4008.42 Aa 5
16.60 ± 2.32
Aa 5
823.61
± 147.79
Aa
83
Fully irrigated
8519.03 ± 2229.90 Aa 8
11.50 ± 3.54
Aa 8
740.79
± 128.59
Ab
92
Water-stressed
6426.27 ± 3905.12 Ba 8
8.38 ± 3.36
Ba 8
767.32
± 232.94
Ba
67
Fully irrigated
2615.73 ± 2081.31 Ba 9
7.00 ± 3.66
Ba 9
373.68
± 207.17
Bb
63
14
F
df
P
F
df
P
F
df
P
Plant species effect
13.81
26 0.001
5.12 26 0.032
37.06
301 < 0.0001
Irrigation treatment effect
0.96
26 0.336
1.16 26 0.291
23.50
301 < 0.0001
a
Number of insects that performed the feeding behavior per irrigation treatment.
b
Number of waveforms events performed per irrigation treatment.
Different lower case letters in columns indicate significant differences between irrigation treatments (α = 0.05).
Different upper case letters in columns indicate significant differences between plant species (α = 0.05).
15
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b Number of waveforms events performed per irrigation treatment.