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DNA barcoding evidence for the North American presence of alfalfa cyst nematode, Heterodera medicaginis

Thomas Powers
Thomas Powers
, 
Andrea Skantar
Andrea Skantar
, 
Tim Harris
Tim Harris
, 
Rebecca Higgins
Rebecca Higgins
, 
Peter Mullin
Peter Mullin
, 
Saad Hafez
Saad Hafez
, 
Zafar Handoo
Zafar Handoo
, 
Tim Todd
Tim Todd
 and 
Kirsten Powers
Kirsten Powers
   | Apr 24, 2019
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Article Category: Arts & Humanities
Published Online: Apr 24, 2019
Page range: 1 - 17
Received: Sep 04, 2018
DOI: https://doi.org/10.21307/jofnem-2019-016
© 2019 Thomas Powers et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Maximum likelihood tree inferred from the COI gene. Each new specimen is represented by an identification number, species name, and location. GenBank accessions are labeled by accession number, name, and the label ‘GB’ (GenBank). Bootstrap support values (%) are labeled in red, the species nodes of H. schachtii, H. medicaginis, and H. glycines are circled in blue.
Maximum likelihood tree inferred from the COI gene. Each new specimen is represented by an identification number, species name, and location. GenBank accessions are labeled by accession number, name, and the label ‘GB’ (GenBank). Bootstrap support values (%) are labeled in red, the species nodes of H. schachtii, H. medicaginis, and H. glycines are circled in blue.

Figure 2

Maximum likelihood tree inferred from the ITS1 gene. Bootstrap support values above 50% are labeled in red. GenBank accession specimen from Russia is boxed in yellow.
Maximum likelihood tree inferred from the ITS1 gene. Bootstrap support values above 50% are labeled in red. GenBank accession specimen from Russia is boxed in yellow.

Figure 3

Phylogenetic relationships of Heterodera species as inferred from analysis of a partial Hsp90 gene using Bayesian inference. Posterior probabilities more than 50% are given next to nodes. Newly obtained sequences are shown in bold.
Phylogenetic relationships of Heterodera species as inferred from analysis of a partial Hsp90 gene using Bayesian inference. Posterior probabilities more than 50% are given next to nodes. Newly obtained sequences are shown in bold.

Figure 4

Heterodera medicaginis juvenile and male specimens. (A–G), juvenile specimens; (H–J), male specimen. (A) NID 7095, entire body; (B) NID 7243, entire body; (C) NID 7095, anterior; (D) NID 7243, anterior; (E) NID 7095, tail; (F) NID 7243, tail; (G) NID 7243, lateral lines; (H) PNID 169028, anterior; (I) PNID 169028, tail; (J) PNID 169028, entire body.
Heterodera medicaginis juvenile and male specimens. (A–G), juvenile specimens; (H–J), male specimen. (A) NID 7095, entire body; (B) NID 7243, entire body; (C) NID 7095, anterior; (D) NID 7243, anterior; (E) NID 7095, tail; (F) NID 7243, tail; (G) NID 7243, lateral lines; (H) PNID 169028, anterior; (I) PNID 169028, tail; (J) PNID 169028, entire body.

Figure 5

(A–D) light micrograph of vulval cones of H. medicaginis showing fenestra (A), bullae (B,D) and underbridge (B,D).
(A–D) light micrograph of vulval cones of H. medicaginis showing fenestra (A), bullae (B,D) and underbridge (B,D).

Figure 6

SEM micrographs of Heterodera medicaginis cysts from Kansas. (A) entire cyst 1, partial view of cyst 2; (B) entire cyst 3; (C) anterior region and excretory pore of cyst 1; (D) anterior region of cyst 3; (E) vulva and anus of cyst 2; (F) vulva of cyst 2 (left) and cyst 1 (right).
SEM micrographs of Heterodera medicaginis cysts from Kansas. (A) entire cyst 1, partial view of cyst 2; (B) entire cyst 3; (C) anterior region and excretory pore of cyst 1; (D) anterior region of cyst 3; (E) vulva and anus of cyst 2; (F) vulva of cyst 2 (left) and cyst 1 (right).

Morphological data on juveniles, all measurements in µm.

Specimen No. Length Stylet Tail Hyaline
1 440 25 – 25
2 421 25 – 21
3 425 25 45 25
4 420 25 50 25
5 421 24 43 22.5
6 420 23 46 23
7 447 25 45 28
8 475 24 52 25.5
9 462 25 45 22.5
10 395 25.5 45 25
11 491 25 – 25.5
12 485 25 50 21
13 413 25 48 25
14 410 26 40 20
15 415 25 45 23
NID 7095 462.5 25 50 27
NID 7096 442.5 24 51 30
NID 7243 477.5 25 52.5 30
NID 7244 455 26 50 25
NID 7245 450 26 49 22.5
NID 7246 425 25 48 27.5
NID 7247 452.5 25 47.5 27.5
NID 8876 472 27 52 28
NID 8877 470 26 50 32
Mean 445.0 25.1 47.8 25.3
SD 27.2 0.8 3.2 3.0
Maximum 491 27 52.5 32
Minimum 395 23 40 20
Gerber and Maas (1982) n = 100 Heterodera medicaginis 462 (417–512) 25 (24–26) 51.8 (41–60) 28.5 (22–33)
Mulvey and Golden (1983) H. glycines 439 ± 6.7 (375–490) 23 ± 0.01 (22–24) 50.4 ± 1.0 (42–59) 26.6 ± 0.7 (20–33)
Mulvey and Golden (1983) H. schachtii 452 ± 49.7 25.6 ± 0.11 48.5 ± 0.73 27.1 ± 0.61

Collection data for specimens used in this study. Specimens 1 to 15 were examined as fixed specimens.

Specimen ID Species Locality Host Marker GenBank Accession No.
1 H. medicaginis Kearny County, Kansas Alfalfa N/A
2 H. medicaginis Kearny County, Kansas Alfalfa N/A
3 H. medicaginis Kearny County, Kansas Alfalfa N/A
4 H. medicaginis Kearny County, Kansas Alfalfa N/A
5 H. medicaginis Kearny County, Kansas Alfalfa N/A
6 H. medicaginis Kearny County, Kansas Alfalfa N/A
7 H. medicaginis Kearny County, Kansas Alfalfa N/A
8 H. medicaginis Kearny County, Kansas Alfalfa N/A
9 H. medicaginis Kearny County, Kansas Alfalfa N/A
10 H. medicaginis Kearny County, Kansas Alfalfa N/A
11 H. medicaginis Kearny County, Kansas Alfalfa N/A
12 H. medicaginis Kearny County, Kansas Alfalfa N/A
13 H. medicaginis Kearny County, Kansas Alfalfa N/A
14 H. medicaginis Kearny County, Kansas Alfalfa N/A
15 H. medicaginis Kearny County, Kansas Alfalfa N/A
P169028 H. medicaginis Kearny County, Kansas Alfalfa 18S AY912048
N838 H. schachtii Goshen County, Wyoming Soybean COI MK093062
N839 H. schachtii Goshen County, Wyoming Soybean COI MK093063
N864 H. schachtii Goshen County, Wyoming Soybean COI MK093064
N4143 Meloidodera sp. Big Thicket National Preserve, Texas Beech COI MK093163
N4178 Meloidodera sp. Big Thicket National Preserve, Texas Water oak COI MK093159
N7083 H. avenae Rio Grande County, Colorado Barley COI MK093164
N7095 H. medicaginis Kearny County, Kansas Alfalfa COI MK093160
N7096 H. medicaginis Kearny County, Kansas Alfalfa COI MK093162
N7243 H. medicaginis Kearny County, Kansas Alfalfa COI ITS1 MK093168 MK093180
N7244 H. medicaginis Kearny County, Kansas Alfalfa COI ITS1 MK093169 MK093181
N7245 H. medicaginis Kearny County, Kansas Alfalfa COI MK093170
N7246 H. medicaginis Kearny County, Kansas Alfalfa COI ITS1 MK093171 MK093182
N7247 H. medicaginis Kearny County, Kansas Alfalfa COI MK093172
N7248 H. trifolii Rio Arriba County, New Mexico Vineyard COI MK093173
N7250 H. trifolii Rio Arriba County, New Mexico Vineyard COI ITS1 MK093174 MK093183
N7251 H. avenae Akershus, Norway Oats COI MK093175
N7253 H. avenae Akershus, Norway Oats COI MK093176
N8306 H. schachtii Platte County, Wyoming Bean COI MK093165
N8876 H. medicaginis Kearny County, Kansas Alfalfa COI MK093166
N8877 H. medicaginis Kearny County, Kansas Alfalfa COI MK093161
N8881 Heterodera sp. Dąbrówka, Poland Peat meadow COI MK093167
P119063 H. medicaginis Carbon County, Montana Alfalfa COI MK093053
P119064 H. schachtii Big Horn County, Montana Alfalfa COI MK093054
P125039 H. schachtii Platte County, Wyoming Bean COI MK093055
P125063 H. schachtii Laramie County, Wyoming Sugar beet COI MK093056
P145076 H. schachtii Platte County, Wyoming Sugar beet COI MK093057
P150049 H. avenae Montana Barley COI MK093058
P150069 Vittatidera zeaphila Tennessee Corn COI MK093060
P164043 H. avenae Montana Barley COI MK093059
P200034 Cactodera sp. Sheridan County, Nebraska Potatoes-wheat COI MK093061
P231089 H. glycines Unknown Soybean COI MK093049
P213017 H. glycines Unknown Soybean COI MK093050
P231088 H. glycines Unknown Soybean COI MK093051
P231091 H. schachtii Unknown Soybean COI MK093052
P243062 H. glycines Richardson County, Nebraska Soybean COI MK093086
P243063 H. glycines Richardson County, Nebraska Soybean COI MK093087
P243064 H. glycines Richardson County, Nebraska Soybean COI MK093088
P243069 H. glycines Seward County, Nebraska Soybean COI MK093077
P243080 H. glycines Lancaster County, Nebraska Soybean COI MK093078
P243083 H. glycines Lancaster County, Nebraska Soybean COI MK093079
P243085 H. glycines Cuming County, Nebraska Soybean COI MK093080
P243086 H. glycines Cuming County, Nebraska Soybean COI MK093081
P243094 H. glycines Nebraska Soybean COI MK093084
P243096 H. glycines Nebraska Soybean COI MK093085
P244003 H. glycines Cedar County, Nebraska Soybean COI MK093095
P244004 H. glycines Cedar County, Nebraska Soybean COI MK093096
P244020 H. glycines Holt County, Nebraska Soybean COI MK093089
P244021 H. glycines Holt County, Nebraska Soybean COI MK093090
P244041 H. glycines Buffalo County, Nebraska Soybean COI MK093093
P244042 H. glycines Buffalo County, Nebraska Soybean COI MK093094
P244060 H. glycines Burt County, Nebraska Soybean COI MK093091
P244062 H. glycines Burt County, Nebraska Soybean COI MK093092
P244065 H. glycines Stanton County, Nebraska Soybean COI MK093082
P244066 H. glycines Stanton County, Nebraska Soybean COI MK093083
P244072 H. glycines Cass County, Nebraska Soybean COI MK093097
P244074 H. glycines Cass County, Nebraska Soybean COI MK093098
P244076 H. schachtii Big Horn County, Montana Pinto bean COI MK093065
P244077 H. schachtii Big Horn County, Montana Pinto bean COI MK093066
P244078 H. schachtii Big Horn County, Montana Black bean COI MK093067
P244079 H. schachtii Big Horn County, Montana Black bean COI MK093068
P244081 H. schachtii Park County, Wyoming Pinto bean COI MK093069
P244082 H. schachtii Park County, Wyoming Pinto bean COI MK093070
P244084 H. schachtii Platte County, Wyoming Pinto bean COI MK093071
P244085 H. schachtii Platte County, Wyoming Pinto bean COI MK093072
P244086 H. schachtii Platte County, Wyoming Pinto bean COI MK093073
P244087 H. schachtii Platte County, Wyoming Pinto bean COI MK093074
P244088 H. schachtii Platte County, Wyoming Pinto bean COI MK093075
P244089 H. schachtii Platte County, Wyoming Pinto bean COI MK093076
P245019 H. glycines Alabama Soybean COI MK093127
P245023 H. glycines Alabama Soybean COI MK093128
P245025 H. glycines Alabama Soybean COI MK093129
P245027 H. glycines Alabama Soybean COI MK093130
P245029 H. glycines Alabama Soybean COI MK093131
P245033 H. glycines Alabama Soybean COI MK093119
P245035 H. glycines Alabama Soybean COI MK093132
P245038 H. glycines Alabama Soybean COI MK093133
P245041 H. glycines Alabama Soybean COI MK093120
P245043 H. glycines Alabama Soybean COI MK093134
P245044 H. glycines Cedar County, Nebraska Soybean COI MK093109
P245046 H. glycines Cedar County, Nebraska Soybean COI MK093110
P245089 H. glycines Oconee County, Georgia Soybean COI MK093111
P245090 H. glycines Oconee County, Georgia Soybean COI MK093112
P245094 H. glycines Burke County, Georgia Soybean COI MK093135
P245095 H. glycines Burke County, Georgia Soybean COI MK093136
P246006 H. glycines Boone County, Missouri Soybean COI MK093121
P246008 H. glycines Boone County, Missouri Soybean COI MK093137
P246010 H. glycines Livingston County, Missouri Soybean COI MK093138
P246014 H. glycines Knox County, Missouri Soybean COI MK093113
P246016 H. glycines Knox County, Missouri Soybean COI MK093114
P246018 H. glycines Atchison County, Missouri Soybean COI MK093139
P246021 H. glycines Atchison County, Missouri Soybean COI MK093122
P246033 H. glycines Fulton County, Ohio Soybean COI MK093123
P246035 H. glycines Fulton County, Ohio Soybean COI MK093140
P246042 H. glycines Sandusky County, Ohio Soybean COI MK093124
P246044 H. glycines Sandusky County, Ohio Soybean COI MK093115
P246047 H. glycines Sandusky County, Ohio Soybean COI MK093116
P247094 H. glycines Lee County, Arkansas Soybean COI MK093141
P247096 H. glycines Lee County, Arkansas Soybean COI MK093142
P247099 H. glycines Lee County, Arkansas Soybean COI MK093143
P248001 H. glycines Lee County, Arkansas Soybean COI MK093144
P248004 H. glycines Washington County, Arkansas Soybean COI MK093145
P248006 H. glycines Washington County, Arkansas Soybean COI MK093146
P248009 H. glycines McLeod County, Minnesota Soybean COI MK093147
P248010 H. glycines McLeod County, Minnesota Soybean COI MK093125
P248013 H. glycines Wilken County, Minnesota Soybean COI MK093117
P248016 H. glycines Wilken County, Minnesota Soybean COI MK093118
P248017 H. glycines Nicollet County, Minnesota Soybean COI MK093148
P248019 H. glycines Nicollet County, Minnesota Soybean COI MK093149
P248023 H. glycines Dodge County, Minnesota Soybean COI MK093099
P248024 H. glycines Dodge County, Minnesota Soybean COI MK093100
P248026 H. glycines Red Lake County, Minnesota Soybean COI MK093101
P248027 H. glycines Red Lake County, Minnesota Soybean COI MK093102
P248029 H. glycines Redwood County, Minnesota Soybean COI MK093150
P248031 H. glycines Redwood County, Minnesota Soybean COI MK093151
P248035 H. glycines Dakota County, Minnesota Soybean COI MK093103
P248036 H. glycines Dakota County, Minnesota Soybean COI MK093104
P248040 H. glycines Waseca County, Minnesota Soybean COI MK093105
P248041 H. glycines Waseca County, Minnesota Soybean COI MK093106
P248058 H. cf. urticae Faulkner County, Arkansas Chickweed COI MK093155
P248059 H. cf. urticae Faulkner County, Arkansas Chickweed COI MK093156
P248060 H. trifolii Washington County, Arkansas Clover COI MK093157
P248061 H. trifolii Washington County, Arkansas Clover COI MK093158
P248066 H. glycines Gentry County, Missouri Soybean COI MK093107
P248068 H. glycines Gentry County, Missouri Soybean COI MK093108
P248071 H. glycines Mercer County, Missouri Soybean COI MK093152
P248073 H. glycines Mercer County, Missouri Soybean COI MK093153
P248086 H. glycines Sussex County, Delaware Soybean COI MK093154
P248087 H. glycines Sussex County, Delaware Soybean COI MK093126
105A13 H. medicaginis Kearny County, Kansas Alfalfa COI MK093177
105A14 H. medicaginis Kearny County, Kansas Alfalfa COI MK093178
105A15 H. medicaginis Kearny County, Kansas Alfalfa COI MK093179
Hsp90_3530 H. medicaginis Kearny County, Kansas Alfalfa Hsp90 MH798843
Hsp90_3531 H. medicaginis Kearny County, Kansas Alfalfa Hsp90 MH798844
Hsp90_3532 H. medicaginis Kearny County, Kansas Alfalfa Hsp90 MH798845
Hsp90_3533 H. medicaginis Kearny County, Kansas Alfalfa Hsp90 MH798846
Hsp90_2996 H. trifolii Alexandria, Egypt Sugar beet Hsp90 MK095224
Hsp90_2997 H. trifolii Alexandria, Egypt Sugar beet Hsp90 MK095225
Hsp90_2994 H. trifolii Alexandria, Egypt Sugar beet Hsp90 MK095222
Hsp90_2995 H. trifolii Alexandria, Egypt Sugar beet Hsp90 MK095223
Hsp90_2998 H. trifolii Alexandria, Egypt Sugar beet Hsp90 MK095226
HetITS105 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093184
HetITS106 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093185
HetITS107 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093186
HetITS108 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093187
HetITS109 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093188
HetITS110 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093189
HetITS111 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093190
HetITS112 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093191
HetITS113 H. medicaginis Kearny County, Kansas Alfalfa ITS MK093192

Host trial for alfalfa cyst nematodes.

Crop
Alfalfa Corn Soybean Tomato
Month 1
Root weight (g) 0.04 0.29 0.21 0.16
Heterodera
Females and cysts 0 0 0 0
Cysts/g 0 0 0 0
Heterodera infective juveniles
Root incubation 70 0 0 0
J2/g 1,750 0 0 0
Meloidogyne J2 infective juveniles
Root incubation 210 4 10 3,100
J2/g 5,250 14 48 19,375
Heterodera/Meloidogyne males
Root incubation 130 0 20 0
Males/g 3,250 0 95 0
Month 2
Root weight (g) 0.69 0.95 0.54 0.23
Heterodera
Females and cysts 52 0 0 0
Cysts/g 75 0 0 0
Heterodera infective juveniles
Root incubation 292 0 0 0
J2/g 423 0 0 0
Meloidogyne J2 infective juveniles
Root incubation 0 0 308 12,040
J2/g 0 0 570 52,348
Heterodera/Meloidogyne males
Root incubation 136 0 56 520
Males/g 197 0 104 2,261
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