Intraspecific variation in phenotypic and phylogenetic features among Pratylenchus penetrans isolates from Wisconsin, USA

Nematodes were recovered from soil samples collected from 11 farms in nine Wisconsin counties for a commodity-sponsored testing program. The samples were randomly selected from a pool of stored samples that were known to contain Pratylenchus males. Nematodes were extracted from root fragments sieved from the soil and incubated in water for 48 hr. One female from the incubated fraction was placed on an excised root culture of ‘IO Chief’ sweet corn (Zea mays L.) grown in Gamborg’s B5 media, with the assumption that insemination occurred in the field. Cultures were maintained in the dark at 24°C and transferred to new Gamborg’s B5 media every five months to increase nematodes. Each population was verified as P. penetrans by morphology, the species-specific primer PPEN (Al-Banna et al., 2004), and matching sequences of the D2/D3 expansion region of 28S rDNA to accessions reported in GenBank. Two additional isolates of P. penetrans in culture for more than 25 years (Portage1 and Portage2), originally extracted from potato, were included in the study for a total of 13 isolates, each named for the county of origin (10 counties total).

Nematodes from at least three Petri dishes were harvested and pooled for morphological characterization, given the reported sensitivity of some features to environmental conditions (Tarte and Mai, 1976a). Fresh specimens were mounted following the protocol of Eisenback (2012), visualized using differential interference contrast optics, and measured and photographed using NIS-Elements AR Software on a Nikon Eclipse Ti-E inverted microscope (Nikon Instruments, Melville, NY). Tail terminus morphology, tail shape, and spermatheca shape were recorded. Data were collected from a minimum of 25 females of each isolate, except for Chippewa2 (n = 18 females), for 26 morphometric and morphological variables. The same morphometrics, except for those that are specific to females, were measured for males of each isolate but only spicule length and gubernaculum are listed in this study. The following indices were derived: V% (anterior end to vulva length/total body length), and a (total body length/maximum body width), b (total body length/esophageal length), c (total body length/tail length), b′ (total body length/distance from anterior end to the end of esophageal glands), and c′ (tail length/body width at anus). Tail shape and tail tip appearance were assigned according to the categories used by Frederick and Tarjan (1989). The coefficient of variation (CV) was computed for each morphological variable by isolate and for pooled data representing all isolates.

Intraspecific variation in the morphometrics among the P. penetrans isolates were evaluated with Hierarchical Agglomerative clustering (HAC) and Canonical Discriminant analysis (CDA) for the following female features: L, V%, ratios of a, b, c, c′, maximum body width, esophageal length, length of anterior end to excretory pore, lip region height, stylet length, DGO, PUS, body width at anus, tail length, vulva to anus distance, lateral incisures, and labial incisures. Based on high correlation coefficients with other features, the b′ ratio, length of head end to posterior gland and body width at vulva were excluded from phenotype analyses. The shape of the tail and annulation of the tail terminus were included as binary variables: subhemispherical tail = 0, not subhemispherical tail = 1; smooth tip = 0, crenate tip = 1.

For HAC, Euclidean distance coefficients were computed on normalized mean values to evaluate the (dis)similarity between each pair of isolates using the ‘dist’ function with the Euclidean method option in the ‘STATS’ R package (R Core Team, 2020). The normalized means were computed by X ′ = X − X min X max − X min , so that all the morphometrics ranged between 0 and 1. HAC was performed using the ‘hclust’ function of the same R package with Ward’s method. The optimum number of clusters was determined using the Elbow and Silhouette methods.

Canonical discriminant analysis (CDA) of the clusters suggested by HAC was performed using the CANDISC procedure in SAS (SAS Institute, Cary, NC) to determine which variables discriminated clusters. Univariate statistics (ANOVA) were also conducted for each morphometric variable to test equality of means among the 13 isolates.

Nematodes from the 13 cultured isolates and 39 field populations collected from 39 farms in 27 Wisconsin counties were characterized molecularly for mitochondrial cytochrome c oxidase subunit 1 (COI) (Fig. 1). The 52 populations were named for the county of origin and multiple populations from the same country were distinguished by numbers and individual specimens by letters (Table 3). Nematode DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). The DNA extracts were stored at −20°C if PCR was not conducted immediately. Five microliters of DNA were suspended in a 25 μl reaction volume composed of 2.5 μl ddH₂O, 2.5 μl of forward and reverse primers, and 12.5 μl of GoTaq Green Master Mix x2 (Promega, Madison, WI). The primer used for COI was forward JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and reverse JB4.5 (5′-TAAAGAAAGAACATAATGAAAATG-3′) (Derycke et al., 2010). PCR cycling conditions were: 94°C for 5 min, 5 cycles of denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec and temperature decreasing with 1°C for each cycle, and extension at 72°C for 30 sec followed by 35 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 30 sec, and a final extension of 10 min at 72°C. Five micro liters of the PCR products were resolved by electrophoresis on 1.0% agarose gel mixed with 2.5 μl of SYBR Safe DNA Stain (Invitrogen, Waltham, WA) at 70 V for 1 hr. The presence of DNA in the PCR products was confirmed by Gel Logic 200 Imaging System (Kodak alaris, Rochester, NY). The crude PCR products were sent to Functional Biosciences (Madison, WI) for purification and Sanger-sequencing.

Figure 1:

The DNA sequences were edited with Finch TV (Geospiza Inc, Seattle, WA), and aligned with MUSCLE in MEGA 7 (Kumar et al., 2016) using default parameters with the gap opening penalty set at 15 and the gap extension penalty at 6.66. Alignments were further adjusted by inspection. A complemented sequence was constructed by overlapping sequence reads with forward and reverse primers for each specimen. Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI) was used to compare the complemented sequences to the GenBank sequence database. Sequences were deposited to GenBank and assigned accession numbers (Table 3).

Redundant COI sequences among multiple individuals sequenced from the same population were dropped, leaving 72 sequences for the haplotype analysis, 13 from cultured isolates, and 59 from field populations. In total, 48 published sequences from populations in other USA states (Ozbayrak et al., 2019) and the populations used in the Janssen et al.’s (2017) study were also included for a total of 120 sequences used in the analyses. The haplotype network was calculated and visualized using TCS network in the software PopART Version 1.7. Geographical origin of the sequences was used as trait information in the analysis.

For phylogenetic analysis, maximum-likelihood (ML) trees were constructed based on the aligned sequences of COI using the default program settings in MEGA 7 with 1000 bootstrap replications. The best DNA model of the nucleotide substitution tool option in MEGA 7 selected the Hasegawa-Kishino-Yano model with a gamma distribution and proportion of invariable sites (HKY + G + I) for COI. The COI sequences of all the Wisconsin populations and CA85, T716, T723, T726, T713, T132 as well as the other species in the Penetrans group and P. crenatus were included in the analysis. Meloidogyne hapla (JX683719) and M. camelliae (KM887147) were included as outgroup taxa per the study by Janssen et al. (2017).

All 13 Wisconsin isolates possessed the diagnostic characters of the species; three lip annuli, round spermatheca filled with sperm, four incisures in the lateral field, and a majority of specimens had a smooth tail tip (Tables 1 and 2). Stylet basal knobs were usually round but sometimes cupped anteriorly. The CV for the pooled data of all isolates was greatest for PUS (24.8%) followed by lip region height (17.4%) and c′ (17.3%). The morphometrics with the smallest variability were V% (1.8%), stylet length (6.0%), and length of head end to the end of the esophageal gland (9.0%). The maximum value for the CV for individual isolates was lower than the pooled CV for body width, lip region height, and stylet length.

Mean morphometric values for the isolates were all within the range reported for P. penetrans by Loof (1960) except three isolates were lower than Loof’s range (5.3-7.9) for b, one was higher than Loof’s range (15-24) for c, and one was lower than Loof’s range (15-17) for stylet length (Tables 1 and 2). All isolates had four lateral incisures but individuals in seven isolates had striae in the middle band in the vicinity of the vulva. Nine isolates had a subhemispherical tail with a smooth tip. Individuals that deviated from this typical tail were detected in four isolates; the frequency of truncate tails ranged from 10 to 30% for three isolates and the frequency of annulated tail tips was ca 10% for three isolates. Males were common in all the isolates.

HAC separated the 13 P. penetrans isolates into four clusters based on the Euclidean distance in canonical space (Fig. 2): (I) Chippewa1 and Marquette1, (II) Chippewa2, Marathon2, Iowa and Portage1, (III) Buffalo and Grant, and (IV) Calumet, Sheboygan, Portage2, Marathon1, and Wood. There was no association between clusters and geographical origins of the isolates. Isolates in HAC clusters I and II had smooth and round tail tips, whereas both isolates in the HAC clusters III and some isolates in the HAC cluster IV (i.e. Calumet and Sheboygan) had a mix of both characters.

Figure 2:

The clusters determined by HAC were confirmed by the CDA analysis (Wilks’ λ < 0.001) (Fig. 3). The means of all the morphometrics, except for esophageal length (P = 0.06), were not equal among the 13 isolates (P < 0.01). The first two canonical variates for the CDA models explained 88% of the variability in morphometrics for clusters based on the HAC groups. The variables with the greatest influence on the discriminant functions (vector loading > 0.5) were lip region height (0.69), maximum body width (−0.61), stylet (−0.55), body width at anus (−0.53), and a (0.50) for the first canonical variate; maximum body width (0.64), total body length (0.61), length from anterior end to excretory pore (0.52) for the second canonical variate; and vulva percentage (0.71) and tail length (0.54) for the third canonical variate. There was reasonable separation of clusters I, II, and III, but the distinction of cluster IV from the other clusters was not resolved (Fig. 3).

Figure 3:

The 402 bp of nucleotide sequence was read without gaps by the COI primer. The pairwise sequence identity between the 13 Wisconsin isolates ranged from 94.2% (Portage1 and Marathon2) to 100% similarity (Table 4). There were 30 single nucleotide polymorphisms (SNPs) on COI sequences among the 13 P. penetrans isolates. The most common mutations were between C and T. Similar mutations were also observed at the same sites among the COI sequences of our field populations and those obtained from GenBank. The largest nucleotide divergence in COI between a Wisconsin population and a population elsewhere was 92.7% for Sheboygan-a and the T716 population (Stramproy) from the Netherlands (Janssen et al., 2017), but the species identification of the Dutch population was not confirmed by a gene in addition to COI. The largest nucleotide divergence in COI with nematodes confirmed to be P. penetrans by 28S rDNA was between Marathon2-a and the V1B population (Meijel) from the Netherlands (Janssen et al., 2017), at 94.5% similarity. There was no variation in amino acid sequences of P. penetrans populations as a result of SNPs on COI.

A total of 120 sequences, 72 from Wisconsin, revealed 21 haplotypes for P. penetrans based on COI (Fig. 4). The Wisconsin populations were assigned to 15 of the 21 haplotypes: three shared with other populations from the USA, seven shared with populations from other countries, and five that were unique to Wisconsin. The most common haplotype in Wisconsin was Pp1, which included 32 Wisconsin populations as well as populations from Nebraska, USA, Colombia, The Netherlands, and Japan (Fig. 4). The second most frequent haplotype in Wisconsin, Pp10, included 19 Wisconsin populations and one population from Idaho, USA. In 17 cases, multiple specimens from the same population were assigned to different haplotype groups (Table 3). No relationship was observed between haplotype and HAC assignment for the 13 Wisconsin isolates.

Figure 4:

The ML tree based on COI validated that the 72 Wisconsin populations were conspecific with P. penetrans (Fig. 5). The bootstrap value supported the assignment of P. penetrans populations to a clade distinguished from the other species in the Penetrans group and P. crenatus. Within the P. penetrans clade, T716, the Dutch population from Janssen et al. (2017), was assigned to a sub-clade that was distant from all others, but it should be noted that species confirmation by 28S rDNA or other genes is not available for that population. Haplotypes Pp1 to Pp4 were distinguished from others and grouped together in one clade, as were Pp6 to Pp8. The evolutionary relationship of other haplotypes was not clearly resolved in the analysis.

Figure 5: