Alfalfa cyst nematode,
According to Subbotin et al. (2010),
During the last five years, there have been unpublished reports of a cyst species reproducing on alfalfa (
The original North American collections of suspect alfalfa cyst specimens were made in western Kansas from alfalfa fields that bordered the Arkansas River near the city of Lakin, Kearny County, KS (Table 1). Some soil was sent to the Kansas State University Diagnostic Laboratory, the University of Idaho Nematology Laboratory, and cysts were sent to the University of Nebraska and the USDA Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL) at Beltsville, Maryland. Additionally, several cysts containing eggs and juveniles were isolated from an alfalfa planting in Carbon County, Montana during a Cooperative Agricultural Pest Survey in 2006 (Table 1).
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 |
|
Kearny County, Kansas | Alfalfa | N/A | |
2 |
|
Kearny County, Kansas | Alfalfa | N/A | |
3 |
|
Kearny County, Kansas | Alfalfa | N/A | |
4 |
|
Kearny County, Kansas | Alfalfa | N/A | |
5 |
|
Kearny County, Kansas | Alfalfa | N/A | |
6 |
|
Kearny County, Kansas | Alfalfa | N/A | |
7 |
|
Kearny County, Kansas | Alfalfa | N/A | |
8 |
|
Kearny County, Kansas | Alfalfa | N/A | |
9 |
|
Kearny County, Kansas | Alfalfa | N/A | |
10 |
|
Kearny County, Kansas | Alfalfa | N/A | |
11 |
|
Kearny County, Kansas | Alfalfa | N/A | |
12 |
|
Kearny County, Kansas | Alfalfa | N/A | |
13 |
|
Kearny County, Kansas | Alfalfa | N/A | |
14 |
|
Kearny County, Kansas | Alfalfa | N/A | |
15 |
|
Kearny County, Kansas | Alfalfa | N/A | |
P169028 |
|
Kearny County, Kansas | Alfalfa | 18S | AY912048 |
N838 |
|
Goshen County, Wyoming | Soybean | COI | MK093062 |
N839 |
|
Goshen County, Wyoming | Soybean | COI | MK093063 |
N864 |
|
Goshen County, Wyoming | Soybean | COI | MK093064 |
N4143 |
|
Big Thicket National Preserve, Texas | Beech | COI | MK093163 |
N4178 |
|
Big Thicket National Preserve, Texas | Water oak | COI | MK093159 |
N7083 |
|
Rio Grande County, Colorado | Barley | COI | MK093164 |
N7095 |
|
Kearny County, Kansas | Alfalfa | COI | MK093160 |
N7096 |
|
Kearny County, Kansas | Alfalfa | COI | MK093162 |
N7243 |
|
Kearny County, Kansas | Alfalfa | COI ITS1 | MK093168 MK093180 |
N7244 |
|
Kearny County, Kansas | Alfalfa | COI ITS1 | MK093169 MK093181 |
N7245 |
|
Kearny County, Kansas | Alfalfa | COI | MK093170 |
N7246 |
|
Kearny County, Kansas | Alfalfa | COI ITS1 | MK093171 MK093182 |
N7247 |
|
Kearny County, Kansas | Alfalfa | COI | MK093172 |
N7248 |
|
Rio Arriba County, New Mexico | Vineyard | COI | MK093173 |
N7250 |
|
Rio Arriba County, New Mexico | Vineyard | COI ITS1 | MK093174 MK093183 |
N7251 |
|
Akershus, Norway | Oats | COI | MK093175 |
N7253 |
|
Akershus, Norway | Oats | COI | MK093176 |
N8306 |
|
Platte County, Wyoming | Bean | COI | MK093165 |
N8876 |
|
Kearny County, Kansas | Alfalfa | COI | MK093166 |
N8877 |
|
Kearny County, Kansas | Alfalfa | COI | MK093161 |
N8881 |
|
Dąbrówka, Poland | Peat meadow | COI | MK093167 |
P119063 |
|
Carbon County, Montana | Alfalfa | COI | MK093053 |
P119064 |
|
Big Horn County, Montana | Alfalfa | COI | MK093054 |
P125039 |
|
Platte County, Wyoming | Bean | COI | MK093055 |
P125063 |
|
Laramie County, Wyoming | Sugar beet | COI | MK093056 |
P145076 |
|
Platte County, Wyoming | Sugar beet | COI | MK093057 |
P150049 |
|
Montana | Barley | COI | MK093058 |
P150069 |
|
Tennessee | Corn | COI | MK093060 |
P164043 |
|
Montana | Barley | COI | MK093059 |
P200034 |
|
Sheridan County, Nebraska | Potatoes-wheat | COI | MK093061 |
P231089 |
|
Unknown | Soybean | COI | MK093049 |
P213017 |
|
Unknown | Soybean | COI | MK093050 |
P231088 |
|
Unknown | Soybean | COI | MK093051 |
P231091 |
|
Unknown | Soybean | COI | MK093052 |
P243062 |
|
Richardson County, Nebraska | Soybean | COI | MK093086 |
P243063 |
|
Richardson County, Nebraska | Soybean | COI | MK093087 |
P243064 |
|
Richardson County, Nebraska | Soybean | COI | MK093088 |
P243069 |
|
Seward County, Nebraska | Soybean | COI | MK093077 |
P243080 |
|
Lancaster County, Nebraska | Soybean | COI | MK093078 |
P243083 |
|
Lancaster County, Nebraska | Soybean | COI | MK093079 |
P243085 |
|
Cuming County, Nebraska | Soybean | COI | MK093080 |
P243086 |
|
Cuming County, Nebraska | Soybean | COI | MK093081 |
P243094 |
|
Nebraska | Soybean | COI | MK093084 |
P243096 |
|
Nebraska | Soybean | COI | MK093085 |
P244003 |
|
Cedar County, Nebraska | Soybean | COI | MK093095 |
P244004 |
|
Cedar County, Nebraska | Soybean | COI | MK093096 |
P244020 |
|
Holt County, Nebraska | Soybean | COI | MK093089 |
P244021 |
|
Holt County, Nebraska | Soybean | COI | MK093090 |
P244041 |
|
Buffalo County, Nebraska | Soybean | COI | MK093093 |
P244042 |
|
Buffalo County, Nebraska | Soybean | COI | MK093094 |
P244060 |
|
Burt County, Nebraska | Soybean | COI | MK093091 |
P244062 |
|
Burt County, Nebraska | Soybean | COI | MK093092 |
P244065 |
|
Stanton County, Nebraska | Soybean | COI | MK093082 |
P244066 |
|
Stanton County, Nebraska | Soybean | COI | MK093083 |
P244072 |
|
Cass County, Nebraska | Soybean | COI | MK093097 |
P244074 |
|
Cass County, Nebraska | Soybean | COI | MK093098 |
P244076 |
|
Big Horn County, Montana | Pinto bean | COI | MK093065 |
P244077 |
|
Big Horn County, Montana | Pinto bean | COI | MK093066 |
P244078 |
|
Big Horn County, Montana | Black bean | COI | MK093067 |
P244079 |
|
Big Horn County, Montana | Black bean | COI | MK093068 |
P244081 |
|
Park County, Wyoming | Pinto bean | COI | MK093069 |
P244082 |
|
Park County, Wyoming | Pinto bean | COI | MK093070 |
P244084 |
|
Platte County, Wyoming | Pinto bean | COI | MK093071 |
P244085 |
|
Platte County, Wyoming | Pinto bean | COI | MK093072 |
P244086 |
|
Platte County, Wyoming | Pinto bean | COI | MK093073 |
P244087 |
|
Platte County, Wyoming | Pinto bean | COI | MK093074 |
P244088 |
|
Platte County, Wyoming | Pinto bean | COI | MK093075 |
P244089 |
|
Platte County, Wyoming | Pinto bean | COI | MK093076 |
P245019 |
|
Alabama | Soybean | COI | MK093127 |
P245023 |
|
Alabama | Soybean | COI | MK093128 |
P245025 |
|
Alabama | Soybean | COI | MK093129 |
P245027 |
|
Alabama | Soybean | COI | MK093130 |
P245029 |
|
Alabama | Soybean | COI | MK093131 |
P245033 |
|
Alabama | Soybean | COI | MK093119 |
P245035 |
|
Alabama | Soybean | COI | MK093132 |
P245038 |
|
Alabama | Soybean | COI | MK093133 |
P245041 |
|
Alabama | Soybean | COI | MK093120 |
P245043 |
|
Alabama | Soybean | COI | MK093134 |
P245044 |
|
Cedar County, Nebraska | Soybean | COI | MK093109 |
P245046 |
|
Cedar County, Nebraska | Soybean | COI | MK093110 |
P245089 |
|
Oconee County, Georgia | Soybean | COI | MK093111 |
P245090 |
|
Oconee County, Georgia | Soybean | COI | MK093112 |
P245094 |
|
Burke County, Georgia | Soybean | COI | MK093135 |
P245095 |
|
Burke County, Georgia | Soybean | COI | MK093136 |
P246006 |
|
Boone County, Missouri | Soybean | COI | MK093121 |
P246008 |
|
Boone County, Missouri | Soybean | COI | MK093137 |
P246010 |
|
Livingston County, Missouri | Soybean | COI | MK093138 |
P246014 |
|
Knox County, Missouri | Soybean | COI | MK093113 |
P246016 |
|
Knox County, Missouri | Soybean | COI | MK093114 |
P246018 |
|
Atchison County, Missouri | Soybean | COI | MK093139 |
P246021 |
|
Atchison County, Missouri | Soybean | COI | MK093122 |
P246033 |
|
Fulton County, Ohio | Soybean | COI | MK093123 |
P246035 |
|
Fulton County, Ohio | Soybean | COI | MK093140 |
P246042 |
|
Sandusky County, Ohio | Soybean | COI | MK093124 |
P246044 |
|
Sandusky County, Ohio | Soybean | COI | MK093115 |
P246047 |
|
Sandusky County, Ohio | Soybean | COI | MK093116 |
P247094 |
|
Lee County, Arkansas | Soybean | COI | MK093141 |
P247096 |
|
Lee County, Arkansas | Soybean | COI | MK093142 |
P247099 |
|
Lee County, Arkansas | Soybean | COI | MK093143 |
P248001 |
|
Lee County, Arkansas | Soybean | COI | MK093144 |
P248004 |
|
Washington County, Arkansas | Soybean | COI | MK093145 |
P248006 |
|
Washington County, Arkansas | Soybean | COI | MK093146 |
P248009 |
|
McLeod County, Minnesota | Soybean | COI | MK093147 |
P248010 |
|
McLeod County, Minnesota | Soybean | COI | MK093125 |
P248013 |
|
Wilken County, Minnesota | Soybean | COI | MK093117 |
P248016 |
|
Wilken County, Minnesota | Soybean | COI | MK093118 |
P248017 |
|
Nicollet County, Minnesota | Soybean | COI | MK093148 |
P248019 |
|
Nicollet County, Minnesota | Soybean | COI | MK093149 |
P248023 |
|
Dodge County, Minnesota | Soybean | COI | MK093099 |
P248024 |
|
Dodge County, Minnesota | Soybean | COI | MK093100 |
P248026 |
|
Red Lake County, Minnesota | Soybean | COI | MK093101 |
P248027 |
|
Red Lake County, Minnesota | Soybean | COI | MK093102 |
P248029 |
|
Redwood County, Minnesota | Soybean | COI | MK093150 |
P248031 |
|
Redwood County, Minnesota | Soybean | COI | MK093151 |
P248035 |
|
Dakota County, Minnesota | Soybean | COI | MK093103 |
P248036 |
|
Dakota County, Minnesota | Soybean | COI | MK093104 |
P248040 |
|
Waseca County, Minnesota | Soybean | COI | MK093105 |
P248041 |
|
Waseca County, Minnesota | Soybean | COI | MK093106 |
P248058 |
|
Faulkner County, Arkansas | Chickweed | COI | MK093155 |
P248059 |
|
Faulkner County, Arkansas | Chickweed | COI | MK093156 |
P248060 |
|
Washington County, Arkansas | Clover | COI | MK093157 |
P248061 |
|
Washington County, Arkansas | Clover | COI | MK093158 |
P248066 |
|
Gentry County, Missouri | Soybean | COI | MK093107 |
P248068 |
|
Gentry County, Missouri | Soybean | COI | MK093108 |
P248071 |
|
Mercer County, Missouri | Soybean | COI | MK093152 |
P248073 |
|
Mercer County, Missouri | Soybean | COI | MK093153 |
P248086 |
|
Sussex County, Delaware | Soybean | COI | MK093154 |
P248087 |
|
Sussex County, Delaware | Soybean | COI | MK093126 |
105A13 |
|
Kearny County, Kansas | Alfalfa | COI | MK093177 |
105A14 |
|
Kearny County, Kansas | Alfalfa | COI | MK093178 |
105A15 |
|
Kearny County, Kansas | Alfalfa | COI | MK093179 |
Hsp90_3530 |
|
Kearny County, Kansas | Alfalfa | Hsp90 | MH798843 |
Hsp90_3531 |
|
Kearny County, Kansas | Alfalfa | Hsp90 | MH798844 |
Hsp90_3532 |
|
Kearny County, Kansas | Alfalfa | Hsp90 | MH798845 |
Hsp90_3533 |
|
Kearny County, Kansas | Alfalfa | Hsp90 | MH798846 |
Hsp90_2996 |
|
Alexandria, Egypt | Sugar beet | Hsp90 | MK095224 |
Hsp90_2997 |
|
Alexandria, Egypt | Sugar beet | Hsp90 | MK095225 |
Hsp90_2994 |
|
Alexandria, Egypt | Sugar beet | Hsp90 | MK095222 |
Hsp90_2995 |
|
Alexandria, Egypt | Sugar beet | Hsp90 | MK095223 |
Hsp90_2998 |
|
Alexandria, Egypt | Sugar beet | Hsp90 | MK095226 |
HetITS105 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093184 |
HetITS106 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093185 |
HetITS107 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093186 |
HetITS108 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093187 |
HetITS109 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093188 |
HetITS110 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093189 |
HetITS111 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093190 |
HetITS112 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093191 |
HetITS113 |
|
Kearny County, Kansas | Alfalfa | ITS | MK093192 |
Preliminary host testing was conducted at Kansas State University using infested field soil containing an estimated 365
Cysts and infective juvenile stages were examined at the USDA MNGDBL and at the University of Nebraska Nematology Laboratory. Select juvenile measurements are presented in Table 2 alongside measurements from Gerber and Maas’s (1982) redescription. Images of juveniles and adult males were taken with a Leica DMLB light microscope with differential interference contrast optics and a Leica DC300 video camera. All juveniles examined at the University of Nebraska were provided an identification number which links specimen images, measurements, and placement on phylogenetic trees. Cysts were prepared for scanning electron microscopy by fixation in a 4% formalin solution followed by a graded series of alcohol to 100% ethyl alcohol prior to critical point drying and coating with gold. Images were obtained on a Hitachi S-3000N scanning electron microscope located in the Morrison Microscopy Core Research Facility at the University of Nebraska.
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)
|
462 (417–512) | 25 (24–26) | 51.8 (41–60) | 28.5 (22–33) |
Mulvey and Golden (1983)
|
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)
|
452 ± 49.7 | 25.6 ± 0.11 | 48.5 ± 0.73 | 27.1 ± 0.61 |
The primers used for amplification of the COI gene region were: COI-F4a-Het-5′-CAGTTATATAATTCTTTTATTACTAGTCATGCATTAATTATRATTTTTTTTYTRGTTATACC-3′. COI-R10b-Het 5′-CCAAAAAAAAAAAAAATCACTATAATCYAAATATTTACGDGG-3′ The sequencing primers were COI-F4a-Het and for the corresponding strand, an internal primer COI-R8-Het-5′-GAAAATGAGCTACCACATAATAAGTATCATGSARAACMACATCCAAACTAGC-3′.
After removal of the primer sequences, amplification products from the
Nematodes amplified at the UNL Nematology Laboratory were individually smashed in 18 µL of sterile H2O with a transparent microfuge micropipette tip on a coverslip, added to a 0.5 mL microfuge tube and stored at −20°C until needed. Amplification conditions were as follows: denaturation at 94°C for 5 min, followed by 45 cycles of denaturation at 94°C for 30 s, annealing at 48.0°C or 50.0°C for 30 s, and extension at 72°C for 90 s with a 0.5°C per second ramp rate to 72°C. A final extension was performed at 72°C for 5 min as described by Powers et al. (2014) and Olson et al. (2017). PCR products were separated and visualized on 1% agarose using 0.5× TBE and stained with ethidium bromide. PCR products of sufficiently high quality were cleaned and sent for sequencing of both strands by the University of California, Davis DNA Sequencing Facility.
Nematodes analyzed in the Beltsville lab were smashed in worm extraction buffer and extracts prepared as described by Skantar et al. (2012). The ITS and 28S rDNA regions were amplified using primers TW81 and AB28, and D2A and D3B, respectively (Skantar et al., 2012). COI was amplified with primers Het-CoxIF and HetCox-1R according to Subbotin et al. (2017). Partial Hsp90 fragments were amplified with primers U288 and L1110 (Skantar et al., 2012). PCR products were cleaned with the Monarch DNA Gel Exraction Kit (NEB, Ipswich, MA). ITS, COI, and Hsp90 amplicons were cloned using the Strataclone PCR Cloning Kit (Agilent, Santa Clara, CA) according to manufacturer’s instructions. Plasmid clones of DNA were prepared with the Monarch Plasmid Miniprep Kit (NEB) and sequenced by Macrogen, Inc.
Nucleotide sequences have been submitted to GenBank and the Barcode of Life Database (BOLD).
Hsp90 sequences obtained for the Kansas population were aligned with partial Hsp90 genomic DNA sequences from other cyst nematode species (new or from GenBank) using the MAFFT algorithm within Geneious 10.2.6. (
ITS1 and COI phylogenetic trees were constructed under maximum likelihood (ML) criteria in MEGA version 6. Sequences were edited using CodonCode Aligner version 8.0.1 (
Host trial results indicated that among the crop species tested, only alfalfa was a suitable host for the Kansas alfalfa
Host trial for alfalfa cyst nematodes.
Crop | ||||
---|---|---|---|---|
Alfalfa | Corn | Soybean | Tomato | |
|
||||
Root weight (g) | 0.04 | 0.29 | 0.21 | 0.16 |
|
||||
Females and cysts | 0 | 0 | 0 | 0 |
Cysts/g | 0 | 0 | 0 | 0 |
|
||||
Root incubation | 70 | 0 | 0 | 0 |
J2/g | 1,750 | 0 | 0 | 0 |
|
||||
Root incubation | 210 | 4 | 10 | 3,100 |
J2/g | 5,250 | 14 | 48 | 19,375 |
|
||||
Root incubation | 130 | 0 | 20 | 0 |
Males/g | 3,250 | 0 | 95 | 0 |
|
||||
Root weight (g) | 0.69 | 0.95 | 0.54 | 0.23 |
|
||||
Females and cysts | 52 | 0 | 0 | 0 |
Cysts/g | 75 | 0 | 0 | 0 |
|
||||
Root incubation | 292 | 0 | 0 | 0 |
J2/g | 423 | 0 | 0 | 0 |
|
||||
Root incubation | 0 | 0 | 308 | 12,040 |
J2/g | 0 | 0 | 570 | 52,348 |
|
||||
Root incubation | 136 | 0 | 56 | 520 |
Males/g | 197 | 0 | 104 | 2,261 |
Figure 1 presents a maximum likelihood tree based on 862 base pairs of the COI mitochondrial gene from 154 specimens of heteroderid species. In total, 13 sequences from isolates collected from soil underneath alfalfa plantings form a well-supported homogeneous group that is a sister group to
The ITS1 tree (Fig. 2) provides less clarity on the distinction between
Four partial Hsp90 sequences were obtained from the Kansas population (Table 1). Although no Hsp90 sequence was available from a reference population of
The 28S sequence from the Kansas population was identical to
Measurements of J2 specimens are reported in Table 2 and illustrated (Fig. 4A–F). The J2 has four lines in the lateral field and a tail with a finely rounded terminus (Fig. 4E–G). The male anterior end, posterior end, and entire body length showing spicules are also depicted (Fig. 4H–J). Morphologically both cysts and juveniles conformed to
A cyst nematode reproducing on alfalfa has been observed along the Arkansas River in western Kansas. A DNA record from a molecular survey from a Montana alfalfa field suggests the distribution may be wider than a single river valley in Kansas. DNA barcoding data by COI sequence rules out the identity of these cyst nematodes on alfalfa as being