Entomopathogenic nematodes (EPNs), which occur naturally in soils, are obligate parasites of soil-inhabiting insects. EPNs were first described in 1923 with the identification of
EPNs are widely distributed throughout the world and have been reported from different kinds of natural and managed habitats and a wide variety of soils (Hominick, 2002; Adams et al., 2006; Adams et al., 2007). The only continent where they have not been found is Antarctica (Griffin et al., 1990). About 95 species of
The rapid killing of the insect host by EPNs and their feasibility of mass production (Ehlers, 2001) has increased interest in searching for and using EPNs in integrated pest management systems (Georgis et al., 2006). Therefore, search for additional potential EPN species is being carried out in different areas on a regular basis (Cimen et al., 2016; Malan et al. 2016; Majić et al., 2018; Godjo et al., 2019; Stock et al., 2019). Indigenous EPNs have been used successfully as biological control agents to suppress various insect populations (Shapiro-Ilan et al., 2002). Indigenous EPNs are more suitable for inundative application against insect pests because of their better adaptation to local environmental conditions, allowing for more persistence and thus greater biological control efficacy. Such native nematodes can be developed as new biological control agents against important insect pests (Burnell and Stock, 2000; Grewal et al., 2002; Lewis et al., 2006). Some previous surveys have focused on finding new EPN species to control important agricultural and horticultural pests under specific conditions (Campos-Herrera and Gutiérrez, 2009). Native EPN species have been isolated from different areas that have showed better heat tolerance (Solomon et al., 2000), foraging ability, virulence (Yu et al., 2010), reproductive potential, or cold adaptation (Ivanova et al., 2001). In addition, the use of exotic EPNs can result in the suppression of native nematodes (Duncan et al., 2003).
Montana is the fourth largest state in the United States with a wide range of habitats, and it can potentially harbor an equally diverse group of EPNs. Until now, no organized survey has been conducted to locate EPNs in this area. Therefore, the objective of this study was to survey EPN diversity in a variety of agricultural habitats in the Golden Triangle Region of Montana and to isolate and identify these EPNs.
Montana is a landlocked state in the northwestern United States and its economy is primarily based on agriculture, including ranching and production of grain (
In the summer of 2018 (May to September), a survey was conducted through parts of the Golden Triangle region. The areas covered during the survey are shown in Table 1. The survey was mainly orientated toward 30 cultivated fields in the region including wheat, lentils, chickpea, peas, alfalfa, and fallow without any crops (Table 1) covering almost all the crops grown in the region. In all the fields, five random 10 to 15 cm deep soil samples were taken within each of the five random plots (8-10 m2) with the help of a hand shovel. Between samples, the shovel was thoroughly rinsed with 70% ethanol to prevent further contamination. Five random samples from each plot were combined to make one composite sample, providing five composite samples from each field. Overall, there were 150 composite samples from 30 fields. The collected samples were then placed in polyethylene bags to prevent water loss and kept in coolers (10°C) during transit to the laboratory. At each field site, data on sampling location, habitat (vegetation), longitude, latitude and elevation were recorded. For each sampling site, a subsample (
Different agricultural field sites surveyed in Montana, USA, for native entomopathogenic nematodes during 2018.
Sampling site | Farmer | Latitude | Longitude | Elevation (m) | Vegetation | Soil texture | Soil pH | Organic Matter (%) | Presence/Absence of EPNs | Re-culture* |
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Pendroy | John Stoltz | N48.04130° | W112.16945° | 4220 | Wheat/Canola | Clay loam | 8.2 | 3.9 | +(3) | No |
Pendroy | Kevin Johnson | N48.04206° | W112.20099° | 4328 | Wheat | Sandy clay loam | 7.9 | 4.4 | +(1) | No |
Choteau | Joe Miller | N47.56785° | W112.13926° | 3906 | Fallow | Clay | 7.8 | 3.2 |
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Choteau | Mike Leys | N47.90238° | W112.3802° |
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Wheat | Sandy clay loam | 8 | 3.3 | +(2) | Yes (1 Steinernematid and 1 Heterorhabditid) |
Valier | John Majerus | N48.18454° | W111.55524° |
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Wheat | Sandy clay loam | 8.1 | 3.4 |
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Conrad | Mark Grub | N48.6334° | W111.53175° |
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Peas | Sandy clay loam | 7 | 2.9 |
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Conrad | Mark Orcutt | N48.16259° | W111.52896° | 3498 | Fallow | Clay loam | 8.1 | 2.3 |
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Conrad | Garett Grub | N47.56786° | W112.13930° |
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Wheat/Vegetables | Sandy clay loam | 7.8 | 2.4 | +(2) | No |
Kalispell | Ron De Yong | N48.1945° | W114.2341° |
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Fallow/Winter wheat | Loam | 8.1 | 3.4 | +(1) | Yes (1 Steinernematid) |
Valier | Jeremy Curry | N48.3032° | W112.4055° | 3749 | Fallow/Wheat | Sandy clay loam | 8.2 | 2.1 | +(2) | No |
Conrad | Zane Drishinski | N48.77764° | W111.8948° | 3508 | Alfalfa | Clay loam | 6.8 | 2.8 |
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Conrad | WTARC | N48.3076° | W111.9255° |
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Fallow/Wheat | Clay loam | 7.8 | 2.9 |
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Conrad | Garman | N48.27808° | W111.92616° | 1116.05 | Alfalfa | Clay loam | 8.1 | 3.7 |
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Choteau (Knees) | A Killion | N48.00729° | W111.36596° | 1081.26 | Chickpea | Clay loam | 8.2 | 2.5 |
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Knees | A Killion | N48.00729° | W111.36596° | 1081.26 | Flax | Clay loam | 7.5 | 3.5 | +(1) | No |
Brady | Winterhood | N47.98468° | W111.84313° | 1112.80 | Fallow/Winter wheat | Sandy clay loam | 7.3 | 1.9 | +(1) | No |
Collins | Chickenhead | N47.95588° | W111.90971° | 1122.68 | Chickpea | Clay | 7.8 | 2.3 | +(1) | No |
Choteau | N47.91965° | W112.04917° | 1177.36 | Hemp | Clay | 8.2 | 2.8 |
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Choteau | Nesbitt | N47.79007° | W111.16589° | 1224.32 | Durum Wheat | Clay loam | 6.6 | 4.4 |
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Dutton | Tims | N47.83529° | W111.53990° | 1144.20 | Lentils | Clay loam | 7.9 | 3.4 |
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Shelby west | John Wigand | N48.56643° | W111.98537° | 1103.57 | Peas | Sandy clay loam | 6.9 | 3.9 |
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Shelby west | John Wigand | N48.57870° | W111.99317° | 1087.12 | Triticale | Sandy loam | 7.6 | 2.6 |
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Shelby West | John Wigand | N48.54374° | W111.99096° | 1071.82 | Fallow | Clay loam | 7.6 | 2.3 |
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Potter Rd Shelby | John Wigand | N48.65339° | W111.96044° | 1057.40 | Fallow | Clay loam | 7.9 | 2.7 |
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Sunburst | Koeye Fauque | N48.82692° | W111.85641° | 1093.77 | Chickpea | Sandy clay loam | 8 | 2.9 | +(1) | No |
Sunburst | Koeye Fauque | N48.82701° | W111.81002° | 1102.46 | Spring wheat | Clay loam | 8.2 | 2.3 |
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Tiber | Duncan | N48.19011° | W111.25206° | 993.03 | Fallow | Clay loam | 8.2 | 2.6 |
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Tiber | Paul Kolstad | N48.17184° | W111.17372° | 960.91 | Fallow | Clay loam | 8.1 | 2.3 |
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Tiber | Wicks | N48.23188° | W111.14511° | 1029.37 | Lentil/Fallow | Clay loam | 7.7 | 2.5 |
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Tiber | Broadhurst | N48.27280° | W111.14571° | 930.70 | Fallow | Clay loam | 8.1 | 2.6 |
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EPNs were isolated from the soil samples using insect baiting techniques (Bedding and Akhurst, 1975). Within a week of soil sampling, a 300 g subsample was transferred to a 500 ml plastic container. Five larvae of
The extracted IJs from different fields were cultured on last instar larvae of
The processed males and IJs were mounted on glass slides and observed for different morphological characters: body length (L), maximum body width (D), tail length (TL), anal body width (ABD), distance from the anterior end to oesophagus (ES), distance from anterior end to excretory pore (EP), and distance from anterior end to nerve ring (NR). In addition, males were observed for spicule length (SP) and Gubernaculum length (GU). The different morphological measurements were recorded using ToupView 3.7 software (Zhejiang, China). According to their morphology, all isolates were placed into different species groups using taxonomic criteria as suggested by Hominick et al. (1997). The morphological characteristics of IJs and males of the EPN isolates were compared statistically by using two-sided
For DNA extraction, pooled EPN IJs of each isolate were macerated with a plastic pestle in 1.5 ml centrifuge tube and genomic DNA was extracted using Qiagen DNeasy® Blood and Tissue kit (Waltham, MA) by following manufacturer’s protocol (Qiagen DNeasy® Blood & Tissue Handbook, 2006). The extracted DNA was concentrated to 20 µl using Eppendorf Vacufuge Plus Vacuum Concentrator (Hamburg, Germany). A part of rDNA comprising the internal transcribed spacer regions (ITS), ITS1 and ITS2 including 5.8 S were sequenced using two sets of primers. Primer set ITS-F (5’-TTGAACCGGGTAAAAGTCG-3 and ITS-R (5’-TTAGTTTCTTTTCCTCCGCT-3’) was used to sequence the entire ITS1, 5.8 S and ITS2 regions (Nadler et al., 2000) while primer set Fnema18S (5’-TTGATTACGTCCCTGCCCTTT-3’) and rDNA1.58 S (rev) (5’-ACGAGCCGAGTGATCCACCG-3’) pair targeted the ITS1 region (Cherry et al., 1997). Each PCR reaction was carried out in a total volume of 30 µl consisting of 9 µl of DNA template, 15 µl of JumpStart™ REDTaq® ReadyMix (Sigma-Aldrich, St. Louis, MO), 2.4 µl of each primer and 1.2 µl of molecular grade water. The PCR conditions included initial denaturation at 94°C for 5 min, 40 cycles of denaturation at 94°C for 30 sec, 40 cycles of annealing at 48°C for 30 sec, 40 cycles of extension at 0.5°C/sec for 90 sec and a final extension at 72°C for 5 min. The PCR products were analyzed for expected DNA band weights on 1% agarose gel run at 150 V for 20 min. PCR products were treated with ExoSAP-IT™ PCR Product Cleanup Reagent (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol to digest excess primers and nucleotides. The products were sequenced bidirectionally with their PCR primers using Bigdye reaction chemistry on an ABI ABI3730xl. Primer sequences were removed from chromatograms and sequences were aligned and edited manually in Geneious Prime 2019.2.1 (
Overall, 150 composite samples (750 single point samples) were collected from the 30 fields in the survey area. Nematodes were recovered from only 15 of the 150 samples. The total percent nematode recovery from samples was 10%. These nematodes can be any kind of nematodes including bacteriophore nematodes. However, out of these 15 samples, we were able to re-culture only three nematode isolates from two fields (one isolate from Ron De Yong (Kalispell); two isolates from Mike Leys (Choteau)) (Table 1). These three isolates were considered EPNs as they were able to reproduce in
The different morphological characteristics of IJs and males for all the EPN isolates are provided in Tables 2 and 3, respectively. Two isolates collected from Mike Leys (Choteau) were found to be different on the basis of morphological data, resulting in two different isolates from the same field. On the basis of morphological characteristics, the isolate from Ron De Yong (Kalispell) was observed to be from the Steinernematidae. However, one isolate from Mike Leys (Choteau) belongs to Heterorhabditidae while the other is from the Steinernematidae.
Morphological characters of 3rd stage infective juveniles for three entomopathogenic nematode isolates from Montana, USA.
Character | Steinernema feltiae 1 | Steinernema feltiae 2 | ANOVA results | Heterorhabditis bacteriophora |
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L | 820.25±11.16 (711-900)a | 807.85±9.60 (743-906)a |
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602.25±9.48 (513-654) |
D | 28.48±0.73 (22-34.4)a | 29.93±0.57 (26-35)a |
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24.74±0.64 (18-31) |
EP | 64.45±1.94 (48-80)a | 55.25±1.72 (43-67)b |
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77.35±2.24 (55-95) |
NR | 72±2.83 (42-91)a | 59.65±2.01 (48-81)b |
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78.2±1.32 (68-90) |
ES | 90.2±2.97 (61-114)a | 87.7±2.02 (48-81)a |
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106.5±3.10 (93-140) |
T | 79.34±1.49 (61-90)a | 80.85±1.09 (74-89)a |
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91.26±4.52 (56-140.3) |
A | 29.15±0.81 (24.58-39.68)a | 27.19±0.6 (23.23-32.78)a |
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24.34±0.60 (14.93-28.5) |
B | 9.31±0.36 (7.45-13.15)a | 9.31±0.25 (7.40-11.13)a |
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5.65±0.18 (3.063-5.52) |
C | 10.40±0.21 (8.67-13.14)a | 10.02±0.16 (8.78-11.19)a |
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6.60±0.29 (2.10-9.16) |
D% | 72.42±2.36 (55.24-87.91)a | 63.62±2.37 (39.81-83.33)a |
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72.63±2.61 (72.29-59.14) |
E% | 81.60±2.63 (63.16-108.11)a | 68.42±2.11 (51.81-86.84)b |
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84.75±5.04 (49.52-98.21) |
Morphological characteristics of males of three entomopathogenic nematode isolates from Montana, USA.
Character | Steinernema feltiae 1 (1st gen.) | Steinernema feltiae 2 (1st gen.) |
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Heterorhabditis bacteriophora (2nd gen.) |
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L | 1570.35±36.84 (1259-1813)a | 1304.8±43.3 (913-1570)b |
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802.1±20.16 (610-920) |
D | 96.93±3.01 (76-130)a | 96.05±3.14 (65-119)a |
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62.3±1.99 (50-78) |
EP | 90.06±2.25 (73-115)a | 93±1.55 (84-109)a |
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77.9±1.17 (70-90) |
NR | 98.35±1.63 (88-113)a | 100±1.18 (88-110)a |
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80.45±1.32 (70-91) |
ES | 145.55±2.80 (121-167)b | 164.55±1.72 (154-180)a |
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110.25±1.97 (97-129) |
T | 41.65±0.75 (36-48)b | 52.75±2.62 (34-72) a |
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61.58±3.59 (41-110) |
ABD | 44.72±1.07 (35-51)b | 62.85±3.00 (45-91)a |
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34.51±0.67 (30-39.4) |
SP | 80.35±1.41 (67-92)a | 71.5±2.21 (58-98)b |
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41±1.26 (32-51) |
GU | 49.95±1.11 (41-59) a | 45.85±0.86 (40-53) b |
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22.95±0.74 (14-28) |
D% | 62.09±1.47 (49.08-72.34)a | 56.56±0.86 (51.14-65.19)a |
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70.90±1.19 (63.2-83.50) |
SW% | 181.27±4.51 (145.65-235.90)a | 117.34±6.81 (63.74-168)a |
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119.83±4.69 (91.37-164.52) |
GS% | 62.74±2.13 (49.40-85.51)a | 66.35±2.16 (51.19-91.38)a |
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56.85±2.61 (41.18-87.5) |
BLASTn analysis showed that the steinernematid recovered from Ron De Yong (Kalispell), and Mike Leys (Choteau) were conspecific to
The second species from Mike Leys (Choteau) was observed to be identical to
The purpose of this survey was to see if EPNs were present in the Golden Triangle area of Montana, and if so, to explore the patterns of their diversity and distribution. Here, we established the occurrence of native EPNs for the first time in Montana, although the recovery percentage of EPNs was very low. The nematodes present in some soil samples caused
The prevalence of EPNs in agro-ecosystems are largely dependent on a number of biotic including host insects, predators such as mites, parasites, pathogens, free living non-EPNs, etc. and abiotic edaphic factors such as temperature, moisture, and UV light (Lewis et al., 2015; Stuart et al., 2015). Mostly, the fields surveyed in this study were cultivated with dryland farming, and low soil moisture levels might have been one of the most important factors for the absence of EPNs and the low recovery rate. When the level of soil moisture is unfavorable, EPNs can go into a resting phase known as “anhydrobiosis” (Grewal, 2000). The negative effect of pesticides used in these agricultural fields may also be responsible for the absence of EPNs.
The measurements of the morphological characters of the three isolates were observed to be similar to those found by Hominick et al. (1997), with some differences in distance from anterior end to excretory pore, nerve ring and oesophagus for IJs and males. The two isolates of
Native EPNs already adapted to local environment are thought to be well suited as inundative biological control agents to suppress different insect pests (Shapiro-Ilan et al., 2002, Lewis et al., 2006). These native nematodes can persist longer in the soil, resulting in better biological control efficacy (Koppenhöfer and Fuzy, 2009). More surveys are needed because of the probability of the presence of additional and more virulent EPN species which can be added to the indigenous gene bank for further research. This will increase our understanding of the diversity and biogeography of EPNs. The new species and strains might be utilized in future ecological and biological control studies against different economically important insect pests in Montana as well as other parts of the world with a similar climate.