Probing behavior of Aphis fabae and Myzus persicae on three species of grapevines with analysis of grapevine leaf anatomy and allelochemicals

Figures & data

Figure 1. Visualization of characteristic aphid probing activities derived from Electrical Penetration Graphs (EPGs) technique. The illustration is composed of representative samples from EPG recordings in the present study. The images for panels a, c and d were obtained from EPG recordings of Aphis fabae on Vitis amurensis and the image for panel b was obtained from EPG recording of Myzus persicae on V. amurensis. “np” – no probing, “C” – pathway activity in apoplast with intracellular punctures, “E1e” – watery salivation into apoplast, “E1” – watery salivation into sieve elements, “F” – derailed stylet activities in apoplast, “G” – sap ingestion from xylem vessels.

Figure 2. Temporal changes in probing behavior of Aphis fabae and Myzus persicae on grapevines Vitis amurensis, Vitis riparia and Vitis vinifera. G = xylem sap ingestion; E1e = watery salivation into apoplast; F = unidentified difficulties in penetration, E1 = watery salivation into sieve elements, E2 = phloem sap ingestion, C = pathway, np = no-probing. “Total” = proportion of all aphid activities during 8-hour EPG monitoring.

Table I. Probing behavior of Aphis fabae on Vitis amurensis, Vitis riparia and Vitis vinifera.

EPG variable^1	Vitis amurensis	Vitis riparia	Vitis vinifera
General aspects	n = 19	n = 21	n = 18
Total duration of no probing (h) ^2	2.7 ± 0.3ab	1.4 ± 0.2b	3.0 ± 0.4a
Total duration of probing in non-phloem tissues C + F + G + E1e (h) ^2	5.8 ± 0.3ab	6.6 ± 0.2b	5.0 ± 0.4a
Total duration of pathway phase C (min) ^2	283.0 ± 15.0	258.0 ± 21.0	238.4 ± 22.2
Total duration of xylem phase G (min) ^2	19.8 ± 8.0ab	45.5 ± 10.1b	20.4 ± 14.7a
Total duration of phase F (min) ^2	36.3 ± 12.4	81.1 ± 24.1	34.7 ± 14.8
Total duration of phase E1e (min) ^2	10.9 ± 5.7	8.8 ± 4.3	6.8 ± 2.3
Total duration of probing in phloem E1+ E2 (min) ^2	0.4 ± 0.2	0.1 ± 0.1	0.0 ± 0.0
Time to the first probe (min) ^2	17.2 ± 9.4	11.6 ± 4.1	5.3 ± 1.7
Number of probes^2	25.1 ± 3.6ab	18.1 ± 1.9b	28.8 ± 2.8a
Number of probes < 2 min	9.4 ± 2.2b	6.0 ± 1.0b	14.6 ± 1.7a
Number of probes >2 min<10 min	5.6 ± 1.3	5.2 ± 0.8	6.6 ± 0.9
Number of probes > 10 min	10.1 ± 0.8ab	6.8 ± 0.6ac	7.6 ± 0.7a
Number of probes with phloem phase^2	0.4 ± 0.1	0.1 ± 0.1	0.1 ± 0.01
Mean duration of a probe (min) ^2	22.1 ± 4.9b	47.3 ± 20.1ab	12.1 ± 1.8a
Time from the beginning of experiment to the first xylem phase G (h)^3	4.0 ± 1.3 n = 7	3.4 ± 0.5 n = 16	n.a. n = 4
Probing in non-phloem tissues before first phloem phase	n = 6	n = 3	n = 1
Time to the first probe (min)^4	3.8 ± 1.5	n.a.	n.a.
Duration of the first probe (min)^4	0.5 ± 0.1	n.a.	n.a.
Number of probes before the first phloem phase^4	25.6 ± 7.4	n.a.	n.a.
Time from the first probe to the first phloem phase (h)^4	5.0 ± 1.1	n.a.	n.a.
Probing in phloem tissues	n = 6	n = 3	n = 1
Duration of the first phloem phase E1 or E1+ E2 (min)^4	0.6 ± 0.1	n.a.	n.a.
Duration of the first phloem salivation period E1 (min) ^4	0.6 ± 0.1	n.a.	n.a.
Duration of the first phloem sap ingestion period E2 (min) ^4	0.0 ± 0.0	n.a.	n.a.
Number of phloem phases^4	1.3 ± 0.2	n.a.	n.a.
Mean duration of phloem phase E1+ E2 (min) ^4	0.9 ± 0.3	n.a.	n.a.
Mean duration of phloem salivation phase E1 (min) ^4	0.9 ± 0.3	n.a.	n.a.
Mean duration of phloem sap ingestion phase E2 (min) ^4	0.0 ± 0.0	n.a.	n.a.
Proportion of phloem phase in total probing (E1+ E2)/(C + F + G + E1e+E1+ E2) ^4	0.003 ± 0.002	n.a.	n.a.
Proportion of salivation in phloem phase E1/(E1+ E2) ^4	1.0 ± 0.0	n.a.	n.a.

¹C = pathway, E1e = watery salivation into apoplast, F = unidentified difficulties in penetration, G = xylem sap ingestion, E1 = watery salivation into sieve elements, E2 = phloem sap ingestion, np = no-probing; ² all replicates (=individual EPG recordings) were included in statistical analysis irrespective of the presence of phloem or xylem phase; ³only replicates that embraced xylem phase were included in statistical analysis; ⁴ only replicates that embraced at least phloem phase E1 were included in statistical analysis; n = number of replicates included in statistical analysis; n.a. = not analyzed statistically due to the low number (fewer than 5) of replicates in which the phloem phase occurred. Values represent means ± SE. Different letters in rows denote statistically significant differences among grapevine species (Kruskal–Wallis test, p < 0.05).

Table II. Probing behavior of Myzus persicae on Vitis amurensis, Vitis riparia and Vitis vinifera.

EPG variable^1	Vitis amurensis	Vitis riparia	Vitis vinifera
General aspects	n = 17	n = 18	n = 19
Total duration of no probing (h)^2	1.9 ± 0.4	1.8 ± 0.4	2.0 ± 0.4
Total duration of probing in non-phloem tissues C + F + G+ E1e (h)^2	6.0 ± 0.4	6.2 ± 0.4	5.9 ± 0.4
Total duration of pathway phase C (min)^2	237.4 ± 15.5	162.8 ± 25.4	220.6 ± 18.7
Total duration of xylem phase G (min)^2	52.4 ± 10.7	52.2 ± 14.0	27.4 ± 5.8
Total duration of phase F (min)^2	34.7 ± 13.0a	96.9 ± 16.8b	39.3 ± 9.8a
Total duration of phase E1e (min)^2	34.9 ± 11.6	60.7 ± 17.4	69.0 ± 21.4
Total duration of probing in phloem E1+ E2 (min)^2	4.8 ± 1.7ab	1.2 ± 0.4ac	2.7 ± 0.6a
Time to the first probe (min)^2	3.3 ± 1.7	8.7 ± 2.8	4.3 ± 1.4
Number of probes^2	39.8 ± 5.8ab	22.9 ± 4.6ac	30.2 ± 3.0a
Number of probes < 2 min	21.1 ± 4.6	9.6 ± 2.1	12.2 ± 1.8
Number of probes >2 min<10 min	10.4 ± 2.1	6.3 ± 1.5	8.5 ± 1.4
Number of probes > 10 min	8.3 ± 0.8	7.0 ± 0.9	9.5 ± 0.8
Number of probes with phloem phase^2	1.5 ± 0.2	0.7 ± 0.3	1.3 ± 0.2
Mean duration of a probe (min)^2	17.9 ± 4.4	26.8 ± 4.0	17.3 ± 3.9
Time from the beginning of experiment to the first xylem phase G (h)^3	2.2 ± 0.4 n = 16	3.1 ± 0.5 n = 17	3.1 ± 0.4 n = 13
Probing in non-phloem tissues before first phloem phase	n = 13	n = 8	n = 14
Time to the first probe (min)^4	4.3 ± 2.1	5.5 ± 4.0	5.1 ± 1.9
Duration of the first probe (min)^4	19.3 ± 9.3b	2.4 ± 0.0ab	1.1 ± 0.6a
Number of probes before the first phloem phase^4	16.7 ± 5.7	18.6 ± 6.9	14.7 ± 0.3
Time from the first probe to the first phloem phase (h)^4	2.6 ± 0.5	3.6 ± 0.8	3.3 ± 0.4
Probing in phloem tissues	n = 13	n = 8	n = 14
Duration of the first phloem phase E1 or E1+ E2 (min)^4	2.5 ± 1.0	1.2 ± 0.4	2.0 ± 0.5
Duration of the first phloem salivation period E1 (min)^4	2.5 ± 1.0	1.2 ± 0.4	2.0 ± 0.5
Duration of the first phloem sap ingestion period E2 (min)^4	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
Number of phloem phases^4	3.2 ± 0.6	2.4 ± 0.5	2.5 ± 0.2
Mean duration of phloem phase E1+ E2 (min)^4	2.0 ± 0.3	1.3 ± 0.4	1.6 ± 0.3
Mean duration of phloem salivation phase E1 (min)^4	2.0 ± 0.3	1.3 ± 0.4	1.6 ± 0.3
Mean duration of phloem sap ingestion phase E2 (min)^4	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
Proportion of phloem phase in total probing (E1+ E2)/(C + F + G + E1e+E1+ E2)^4	0.02 ± 0.00	0.01 ± 0.00	0.01 ± 0.00
Proportion of salivation in phloem phase E1/(E1+ E2)^4	1.0 ± 0.0	1.0 ± 0.0	1.0 ± 0.0

¹C = pathway, E1e = watery salivation into apoplast, F = unidentified difficulties in penetration, G = xylem sap ingestion, E1 = watery salivation into sieve elements, E2 = phloem sap ingestion, np = no-probing; ² all replicates (=individual EPG recordings) were included in statistical analysis irrespective of the presence of phloem or xylem phase; ³only replicates that embraced xylem phase were included in statistical analysis; ⁴ only replicates that embraced at least phloem phase E1 were included in statistical analysis; n = number of replicates included in statistical analysis;. Values represent means ± SE. Different letters in rows denote statistically significant differences among grapevine species (Kruskal–Wallis test, p < 0.05).

Figure 3. Proportion of Aphis fabae and Myzus persicae that reached vascular tissues, phloem and xylem, in the leaves of Vitis amurensis, Vitis riparia and Vitis vinifera.

Figure 4. Proportion of phenolic compounds analyzed in grapevines Vitis amurensis, V. riparia and V. vinifera.

Figure 5. Representative samples of transverse sections of grapevine leaves at main veins of the leaves of Vitis amurensis, V. riparia and V. vinifera observed under light microscope Carl Zeiss Axio Imager 2 coupled with Zeiss AxioCam ERc 5s and ZEN Lite computer programme (400x magnification). 1 – adaxial epidermis; 2 – collenchyma; 3 – parenchyma; 4 – vascular bundle sheath; 5 – phloem; 6 – xylem.

Table III. Anatomical measurements of leaves of Vitis amurensis, Vitis riparia and Vitis vinifera.

	Vitis amurensis	Vitis riparia	Vitis vinifera
Epidermis
Proximal	5.1 ± 0.4	6.1 ± 0.2 a	5.7 ± 0.3
Distal	5.0 ± 0.2 A	8.1 ± 0.2 b B	5.6 ± 0.3 A
Epidermis-Phloem
Proximal	80.9 ± 5.0 a B	86.6 ± 6.2 B	62.3 ± 3.4 A
Distal	109.3 ± 9.1 b B	68.4 ± 3.7 A	58.1 ± 2.9 A
Pathway
Proximal	275.6 ± 40.9	279.0 ± 37.8 a	181.6 ± 17.7
Distal	384.7 ± 54.1 B	156.7 ± 12.0 b A	146.8 ± 15.7 A

“Epidermis” = abaxial epidermal thickness (μm); “Epidermis-phloem” = distance from leaf surface to phloem (= thickness of mesophyll and epidermis, i.e., the distance between abaxial leaf surface and the closest phloem vessel; in μm); “Pathway” = simulation of the shortest aphid stylet penetration pathway from abaxial leaf surface to phloem (μm²). Values represent means ± SE; n = 9. Different small letters in columns indicate significant differences between proximal and distal parts of the leaf within a grapevine species (Mann-Whitney U-test, p < 0.05); different capital letters in rows indicate significant differences among grapevine species (Kruskal–Wallis test, p < 0.05)