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Morphological and Molecular Analysis of Two Mycophagous Nematodes, Aphelenchoides bicaudatus and A. rutgersi (Nematoda: Aphelenchoididae) from Florida Strawberry

Clemen J. Oliveira
Clemen J. Oliveira
, 
Sergei A. Subbotin
Sergei A. Subbotin
, 
Johan A. Desager
Johan A. Desager
, 
Paul Dahlin
Paul Dahlin
, 
Silvia Vau
Silvia Vau
 e 
Renato N. Inserra
Renato N. Inserra
   | 05 lug 2024
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Article Category: Research Paper
Pubblicato online: 05 lug 2024
Pagine: -
Ricevuto: 29 apr 2023
DOI: https://doi.org/10.2478/jofnem-2024-0021
Parole chiave
© 2024 Clemen J. Oliveira et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1.

Photomicrographs of Aphelenchoides bicaudatus female from Florida. (A) Anterior body showing the (E) excretory pore and the (PO) pharyngeal overlap. (B) Vulvar region showing (V) vulva and (PB) postuterine branch. (C) Lateral field (LF) marked by two outer visible incisures. The inner incisure is not discernible. (D), (E) Posterior body showing (A) anus and shape variations of the bifurcate tail terminus.
Photomicrographs of Aphelenchoides bicaudatus female from Florida. (A) Anterior body showing the (E) excretory pore and the (PO) pharyngeal overlap. (B) Vulvar region showing (V) vulva and (PB) postuterine branch. (C) Lateral field (LF) marked by two outer visible incisures. The inner incisure is not discernible. (D), (E) Posterior body showing (A) anus and shape variations of the bifurcate tail terminus.

Figure 2.

Photomicrographs of Aphelenchoides rutgersi female and male from Florida. (A) Female anterior body. (B) Pharyngeal region of female showing (M) median bulb and (PO) pharyngeal overlap. (C) Lateral field (LF) marked by three incisures. (D) Vulvar region showing (O) ovary with oocytes, (V) vulva, and portion of the (PB) post-vulvar uterine branch. (E) Posterior body of female showing (A) anus (A) and (N) the tail terminus with a ventral fine mucro. (F): Posterior body of male showing (S) spicules.
Photomicrographs of Aphelenchoides rutgersi female and male from Florida. (A) Female anterior body. (B) Pharyngeal region of female showing (M) median bulb and (PO) pharyngeal overlap. (C) Lateral field (LF) marked by three incisures. (D) Vulvar region showing (O) ovary with oocytes, (V) vulva, and portion of the (PB) post-vulvar uterine branch. (E) Posterior body of female showing (A) anus (A) and (N) the tail terminus with a ventral fine mucro. (F): Posterior body of male showing (S) spicules.

Figure 3.

Phylogenetic relationships of A. bicaudatus and A. rutgersi with some Aphelenchoides species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the 18S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to, or more than, 0.7 are given for appropriate clades. New sequences are indicated in bold letters. * - identified as Aphelenchoides sp. in the GenBank.
Phylogenetic relationships of A. bicaudatus and A. rutgersi with some Aphelenchoides species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the 18S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to, or more than, 0.7 are given for appropriate clades. New sequences are indicated in bold letters. * - identified as Aphelenchoides sp. in the GenBank.

Figure 4.

Phylogenetic relationships of A. bicaudatus and A. rutgersi with some Aphelenchoides species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the D2-D3 of 28S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to, or more than, 0.7 are given for appropriate clades. New sequences are indicated in bold letters. * - identified as Aphelenchoides sp. in the GenBank. ** - identified as an uncultured fungus in the GenBank.
Phylogenetic relationships of A. bicaudatus and A. rutgersi with some Aphelenchoides species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the D2-D3 of 28S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to, or more than, 0.7 are given for appropriate clades. New sequences are indicated in bold letters. * - identified as Aphelenchoides sp. in the GenBank. ** - identified as an uncultured fungus in the GenBank.

Figure 5.

Phylogenetic relationships of A. bicaudatus and A. rutgersi with some Aphelenchoides species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the COI gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to, or more than, 0.7 are given for appropriate clades. New sequences are indicated in bold letters.
Phylogenetic relationships of A. bicaudatus and A. rutgersi with some Aphelenchoides species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the COI gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to, or more than, 0.7 are given for appropriate clades. New sequences are indicated in bold letters.

Figure 6.

Symptoms induced by Aphelenchoides bicaudatus on soybean leaves. (A) Seedling showing discoloration and necrosis on the upper surface of the blade adjacent to the petiole 3 d after inoculation of the nematode using pieces of filter paper attached to the leaf Blade. (B) Closeup of the lesion delimited by the veins of the leaflet showing desiccated areas and dark tissues along the veins 14 d after the inoculation of the nematode.
Symptoms induced by Aphelenchoides bicaudatus on soybean leaves. (A) Seedling showing discoloration and necrosis on the upper surface of the blade adjacent to the petiole 3 d after inoculation of the nematode using pieces of filter paper attached to the leaf Blade. (B) Closeup of the lesion delimited by the veins of the leaflet showing desiccated areas and dark tissues along the veins 14 d after the inoculation of the nematode.

Figure 7.

Aphelenchoides bicaudatus inside soybean leaf tissues. (A) A nematode specimen tunneling the mesophyll 7 d after the inoculation of 300 specimens delivered with a piece of filter paper attached to the leaf blade. (B) Posterior body of a specimen showing the bifurcate tail terminus. (C) Anterior body of a specimen showing the stylet. (Scale bar = 24 μm).
Aphelenchoides bicaudatus inside soybean leaf tissues. (A) A nematode specimen tunneling the mesophyll 7 d after the inoculation of 300 specimens delivered with a piece of filter paper attached to the leaf blade. (B) Posterior body of a specimen showing the bifurcate tail terminus. (C) Anterior body of a specimen showing the stylet. (Scale bar = 24 μm).

Populations of Aphelenchoides characterized in the present study.

Species Location Host Sample code GenBank accession number Source
SSU LSU COI
A. bicaudatus Wimauma, Florida, USA Fragaria × ananassa N18-1001-3 OK644201 OK644281 OK644199 C. Oliveira
OK644282
A. rutgersi Wimauma, Florida, USA Fragaria × ananassa N18-00206 OK644202 OK644283 OK644295 C. Oliveira
OK644203

Morphometrics of live females of a Florida population of Aphelenchoides bicaudatus from strawberry reared on Monilinia fructicola compared to those in the original (Imamura, 1931) and subsequent re-descriptions by Siddiqui & Taylor (1967), Jen et al. (2012), Kim et al. (2016), Israr et al. (2017) and Shokoohi and Moyo (2022).a

Population Substrate Florida Monilinia fructicola Japan Paddy field Illinois Pyrenochaeta terrestris Taiwan Alternaria citri South Korea Botrytis cinerea Pakistan Raphanus sativus South Africa Pennisetum clandestinum
Reference Present paper N18-01001-3 Imamura (1931) Siddiqui and Taylor (1967) Jen et al. (2012) Kim et al. (2016) Israr et al. (2017) Shokoohi and Moyo (2022)
Character n 10♀♀ 18♀♀ 50♀♀ 50♀♀ 7♀♀ 2♀♀ 8♀♀
L 494.1 ± 32.0 499 ± 67.9 517.9 ± 3.8 455.0 ± 64.5
(454.8–552.4) (380.0–470.0) (410.0–550.0) (376–637) (513.6–522.6) (360.0–360.0) (409.0–529.0)
A 31.2 ± 3.6 33.0 ± 2.4 28.3 ± 0.5 29.1 ± 2.0
(27.6–41.1) (31.3–31.7) (25–31) (27.0–38.6) (27.7–28.8) (30.1–32.7) (27.1–31.1)
B 8.0 ± 0.5 9.0 ± 0.7 7.3 ± 0.0 4.6 ± 0.5
(7.2–9.0) (6.8–8.4) (7.3–9.6) (7.5–10.0) (7.3–7.4) (8.8–7.2) (4.1–5.1)
b’ 4.1 ± 0.2 - - 5.1 ± 0.7 - -
(3.7–4.6) (3.6–7.9) (5.6–5.8)
C 13.2 ± 0.9 11.9 ± 0.9 11.3 ± 0.5 16.3 ± 2.0
(11.9–14.8) (9.4–12.6) (9.8–13.7) (10.1–14.8) (10.7–11.9) (11.3–12.0) (14.1–17.8)
c’ 4.4 ± 0.5 - - 5.4 ± 0.5 4.6 ± 0.1 3.2 ± 0.6
(3.7–5.3) (4.1–7.1) (4.4–4.8) (2.9–3.7) (2.6–3.6)
V 68.7 ± 1.5 68.5 ± 1.2 66.0 ± 0.2 68.8 ± 1.6
(65.8–71.1) (61.7–90.2) (65.0–70.0) (64.9–71.8) (65.7–66.4) (66.8–67.2) (67.0–71.0)
OV 27.8 ± 2.0 - - 15.2 ± 2.7 - -
(23.9–30.9) (11.0–22.0) (12.0–12.5)
Max. body diameter 15.9 ± 1.3 - - - - 15.6 ± 1.5
(13.5–17.8) (25.0–26.2) (14.0–17.0)
Body diameter at anus 8.6 ± 0.8 - - - - 8.8 ± 0.7
(7.2–9.9) (11.8) (8.0–9.0)
Anterior genital tract length 136.9 ± 10.4 - - - - 179.3 ± 18.0
(121.7–150.1) (84.0–95.0) (167.0–200.0)
Lip region width 4.9 ± 0.3 - - - 5.7 ± 0.1
(4.5–5.5) (5.0–5.2) (4.0–4.3) (5.6–5.8)
Lip region height 2.8 ± 0.2 - - - 2.8 ± 0.9
(2.4–3.0) (2.5–2.9) (2.0–2.0) (2.0–4.0)
Stylet length 10.9 ± 0.4 - 10.4 ± 0.6 11.2 ± 0.5 11.1 ± 1.7
(10.3–11.8) (10–12) (9.0–12.0) (10.4–11.7) (10.0–11.0) (10.0–13.0)
Stylet cone 5.1 ± 0.3 - - - - - 4.7 ±0.3
(4.5–5.8) (4.5–5.0)
Stylet knob height 1.4 ± 0.2 - - - - - -
(1.2-1.7)
Stylet knob width 1.9 ± 0.3 - - - - - -
(1.2–2.3)
Median bulb length 12.1 ± 0.7 - - - 11.2 ± 0.7
(10.9–13.3) (12.8–12.9) (10.0–10.0) (11.0–12.0)
Median bulb width 9.6 ± 0.8 - - - 9.0 ±0.8
(7.9–11.1) (8.6–8.9) (7.0–8.0) (8.5–10.0)
Median bulb valve length 4.1 ± 0.4 - - - - - -
(3.6–4.9)
Median bulb valve width 2.9 ± 0.2 - - - - - -
(2.5–3.2)
Pharynx length 62.1 ± 2.7 - - - - 88.3 ± 11.2
(59.0–68.7) (90.0–92.0) (76.0–98.0)
Pharyngeal overlap 57.0 ± 2.1 - - - - -
(53.0–59.7) (50.0–75.0)
Ant. end to pharyngeal gland lobe 118.8 ± 3.3 - - - - - -
(4.5–5.8)
Anterior end to excretory pore 66.9 ± 3.0 - - - - 61.3 ± 3.2
(62.3–71.2) (50.0–51.0) (60.0–65.0)
Postuterine sac (PUS) 22.3 ± 1.9 - - - - 47.0 ± 2.6
(18.0–24.2) (22.0–24.0) (45.0–50.0)
Vulva anus distance (VA) 123.5 ± 10.3 - - - - -
(105.9–137.6) (84.0–85.0)
Ant. end to vulva 349.3 ± 22.3 - - - - -
(311.0–392.7) (242.0–248.0)
Post end to vulva 144.8 ± 26.5 - - - - - -
(89.3–170.4)
Tail length 37.4 ± 0.8 - 45.9 ± 2.5 28.0 ± 3.6
(36.6–38.6) (41.8) (34–42) (43.0–48.8) (30.0–31.0) (24.0–31.0)
Body width at vulva (BWV) 14.5 ± 0.8 - - - - - -
(13.1–15.8)
PUS/L 4.5 ± 0.4 - - - - -
(3.7–5.2) (6.1–6.6)
PUS/BWV 1.6 ± 0.2 - - - - - -
(1.1–1.8)
PUS/VA 18.1 ± 1.1 - - 18.9 ± 4.5 - -
(16.0–20.5) (9.2–33.8) (22.4–24.0)
Lateral field width 2.7 ± 0.4 - - - - -
(2.1–3.4) (2.2)

Morphometrics of live females of a Florida populations of Aphelenchoides rutgersi from strawberry and reared on Monilinia fructicola compared to those in the original description by Hooper and Myers (1971) and populations from Pakistan and South Africa reported by Erum and Shahina (2010) and Girgan et al. (2018), respectively.a

Population Substrate Florida Monilinia fructicola Florida Pyrenochaeta terrestris Florida Botrytis cinerea for 2 weeks Florida B. cinerea for 7 weeks Pakistan Triticum sp. South Africa Aristida canescens seeds
Reference Present study (N18-00206) Hooper and Myers (1971) Hooper and Myers (1971) Hooper and Myers (1971) Erum and Shahina (2010) Girgan at al. (2018)
Character n 20♀♀ 20♀♀ 20♀♀ 20♀♀ 12♀♀ 3♀♀
L 507.6 ± 71.0 535.0 ± 20 480.0 ± 25 360.0 ± 20 440 ± 30 556.0 ± 22.6
(371.2–614.7) (500.0–570.0) (430.0–530.0) (320.0–405.0) (370.0–500.0) (542.0–582.0)
A 29 ± 2 27.0 ± 1.8 25.0 ± 1.7 27.0 ± 1.7 26.7 ± 1.4 34.1 ± 0.8
(25.8–33.5) (23–30) (23–29) (24–31) (24.0–31.7) (33.5–34.9)
B 8.4 ± 1.0 10.7 ± 0.4 9.3 ± 0.4 7.60 ± 0.16 5.7 ± 1.3 4.6 ± 0.2
(6.5–10.0) (9.7–11.6) (8.3–10.1) (6.7–8.9) (4.4–8.4) (4.4–4.8)
b’ 4.0 ± 0.6 5.0 ± 0.5 4.1 ± 0.4 3.5 ± 0.3 4.4 ± 0.8 -
(3.0–5.2) (4.2–6.2) (3.6–4.6) (3.1–4.0) (4.1–5.0)
C 15.4 ± 1.7 16.9 ± 0.6 16.3 ± 1.0 14.1 ± 0.8 8.0 ±0.4 15.8 ± 0.5
(12.9–18.9) (15.9–18.1) (15.0–18.7) (12.4–15.5) (7.2–8.6) (15.5–16.4)
c’ 3.4 ± 0.4 3.0 ± 0.2 2.9 ± 0.1 3.3 ± 0.2 5.7 ± 0.4 3.6 ± 0.3
(2.5–4.0) (2.6–3.4) (2.6–3.2) (2.8–4.0) (4.8–6.4) (3.3–3.9)
V 70.3 ± 1.0 71.0 ± 1.0 72.0 ± 0.9 70.0 ± 0.7 64.5 ± 0.9 69.0 ± 0.8
(68.6–72.0) (69.0–72.0) (70.0–74.0) (69.0–72.0) (63.3–65.5) (69.0–70.0)
Max. body diameter 17.6 ± 3.1 - - - - 16.0 ± 0.9
(12.8–22.7) (16.0–17.0)
Body diameter at anus or cloacal opening 10.0 ± 1.4 - - - - 10.0 ± 0.9
(7.4–13.3) (9.0–11.0)
Body diameter at vulva (BDV) 18.1 ± 2.3 - - - - 16.0 ± 0.8
(15–23) (15.017.0)
OV or Testis/L 24.8 ± 6.4 60.0 ± 7.1 50.0 ± 4.6 24.0 ± 2.8 - -
(15.4–40.9) (50–77) (42–60) (18–29)
Anterior genital tract length 127.2 ± 44.0 - - - - 220.0 ± 5.6
(70.0–242.5) (215.0–226.0)
Lip region width 6.3 ± 0.5 - - - 5.6 ± 0.4
(5.0–6.9) (4.8–5.6) (5.0–6.0)
Lip region height 2.9 ± 0.2 - - - 3.2 ± 0.5
(2.5–3.0) (2.4–3.2) (3.0–4.0)
Stylet length 11.0 ± 0.5 - - 10.7 ± 0.6 11.0 ± 0.4
(10.1–11.9) (10.0) (10.0–12.0) (11.0–12.0)
Stylet cone 5.4 ± 0.4 - - - - -
(4.8–6.0)
Stylet knob height 1.5 ± 0.2 - - - - -
(1.2–1.9)
Stylet knob width 2.3 ± 0.3 - - - - -
(1.9-2.8)
Median bulb length 13.7 ± 0.7 - - - 12.0 ±0.5
(12.3–14.9) (12.8–13.6) (12.0–13.0)
Median bulb width 10.3 ± 0.7 - - - 10.0 ± 0.5
(9.0-11.9) (9.6–10.4) (9.0–10.0)
Median bulb valve length 4.0 ± 0.4 - - - - -
(3.1–4.5)
Median bulb valve width 3.0 ± 0.2 - - - - -
(2.5–3.4)
Pharynx length 60.4 ± 2.1 44 ± 2 46 ± 2 27.0 ± 1.7 -
(54.4–66.3) (42–47) (42–49) (24–31) (58.0)
Pharyngeal overlap 67.5 ± 12.5 - - - - -
(45.5–95.8)
Ant. end to pharyngeal gland lobe 127.8 ± 13.7 - - - - 122.0 ± 1.5
(99.9–162.1) (121.0–124.0)
Anterior end to excretory pore 66.6 ± 5.4 - - - - 76.0 ±1.9
(58.4–76.2) (75.0–76.0)
Postuterine sac 26.1 ± 4.5 - - - 42.6 ±2.2 40.0 ± 4.1
(15.8–41.0) (36.0–55.0) (37.0–43.0)
Vulva anus distance 117.7 ± 19.2 - - - - -
(76.2–149.4)
Ant. end to vulva 356.9 ± 50.3 - - - 386.0 ± 13.6 -
(266.3–437.5) (374.0 401.0)
Post end to vulva 150.7 ± 21.7 - - - - -
(104.9–191.9)
Tail length 33.0 ± 3.6 - - 35.0 ± 2.1 -
(26.7–42.5) (30.5) (33.0–37.0)
Spermatheca length 14.1 ± 3.9 - - - - -
(9–21)
Spermatheca width 9.9 ± 1.6 - - - - -
(8–13)
PUS/VA 22.1 ± 3.8 - - - - -
(16.1–27.5)
Lateral field width 3.0 ± 0.2 - - - - -
(2.5–3.4)
PUS/L 5.1 ± 0.8 - - - - -
(3.8–6.4)
PUS/BDV 1.5 ± 0.2 - - - - -
(1.0–1.9)
Spikes 1 1 1 - 1 -

Morphometrics of live males of a Florida population of Aphelenchoides rutgersi from strawberry reared on Monilinia fructicola compared to those in the original description by Hooper and Myers (1971) and a population from Pakistan described by Erum and Shahina (2010).a

Population Substrate Florida Monilinia fructicola Florida Botrytis cinerea Pakistan Triticum sp.
Reference Present study N18-00206 Hooper and Myers (1971) Erum and Shahina (2010)
Character n 2♂♂ 20♂♂ 7♂♂
L 538.8 ± 20.5 440.0 ± 35 400.0 ± 30.0
(518.3−559.2) (380.0−490.0) (360.0−470.0)
A 29.7 ± 0.3 29.0 ± 2.8 28.7 ± 2.5
(29.4−30.0) (21−32) (24.0−31.7)
B 8.9 ± 0.4 8.7 ± 0.9 5.3 ± 1.3
(8.5−9.3) (7.1−11.0) (3.9−7.2)
b’ 4.0 ± 0.1 4.0 ± 0.1 4.5 ± 1.0
(4.0−4.1) (3.6−4.4) (4.2−5.5)
C 15.9 ± 1.2 14.3 ± 0.8 13.7 ± 1.6
(14.7−17.2) (13.0−15.4) (10.6−16.0)
c’ 2.6 ± 0.3 2.9 ± 0.2 2.7 ± 0.3
(2.3−2.9) (2.4−3.3) (2.4−3.1)
Max. body diameter 18.2 ± 0.9 - -
(17.3−19.0)
Body diameter at cloacal opening 13 ± 1 - -
(12−14)
Testis 240.5 ± 8.5 - -
(232−249)
Testis/L% 44.7 ± 0.1 72.0 ± 6.2 -
(44.5−44.8) (58.0−78.0)
Lip region width 6.5 ± 0.2 - -
(6.3−6.6)
Lip region height 3 - -
Stylet length 11.9 10.0 ± 1 (9.5−11.0) 10.6 ± 0.4
(10.0−11.2)
Stylet cone 6 - -
(5.9−6.0)
Stylet knob width 2.4 ± 0.1 - -
(2.3−2.4)
Stylet knob height 1.8 ± 0.1 - -
(1.7−1.8)
Median bulb width 10.0 ± 0.5 - -
(9.5−10.5)
Median bulb length 14.2 ± 0.7 - -
(13.5−14.8)
Median bulb valve length 4.5 - -
Median bulb s valve width 3 - -
Pharynx length 60.6 ± 0.2 45 ± 3 -
(60.4−60.8) (38.0−51.0)
Pharyngeal overlap 72.8 ± 3.5 - -
(69.3−76.2)
Ant. end to pharyngeal gland lobe 133.7 ± 2.9 - -
(130.8−136.6)
Anterior end to excretory pore 67.8 ± 3.5 - -
(64.3−71.2)
Tail length 33.9 ± 1.3 - -
(32.6−35.2)
Spicule length 18.2 ± 0.6 15.5 ± 1.0 14.2 ± 1.7
(16.6−19.8) (14−17) (13.0−17.0)
Lateral field width 3 - -
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