First Report of the Spiral Nematode Helicotylenchus microlobus Infecting Soybean in North Dakota

摘要

Spiral nematodes (Helicotylenchus spp.) are common plant-parasitic nematodes in ?elds of many crops.In June 2015, two soil samples were collected from a soybean ?eld in Richland County, ND.Nematodes were extracted from soil using the sugar centrifugal ?otation method (Jenkins, 1964).Plant-parasitic nematodes were identi?ed to genus based on morphological features and counted.Both samples contained spiral nematodes from 1,500 to 3,300 per kilogram of soil.In June and August 2016, 10 soil samples were collected from the same ?eld.Nematodes were extracted, and nine of the samples had spiral nematodes ranging from 125 to 3,065 per kilogram of soil.One soil sample with 1,500 spiral nematodes per kilogram was used to inoculate two soybean cultivars Sheyenne and Barnes each in four replicates.After 15 wk of growth at 228C in a greenhouse room, the population of spiral nematodes was found to have increased greatly.The ?nal density was 9,300 6 1,701 spiral nematodes per kilogram of soil for Sheyenne and9,45162,751forBarnes.ThereproductivefactorinSheyenneandBarneswas6.2and6.3, respectively,indicatingthat this spiral nematode infects and reproduces well on these two soybean cultivars.Infected soybean roots had small brown lesions on the surface.Individual spiral nematodes were handpicked and examined morphologically and molecularly for species identi?cation.Morphological measurements of adult females (n = 15) included body length (mean = 708.5 mm, range = 600.0–812.0 mm), stylet (27.6, 26.0–29.0), body width (28.3, 25.0–33.0), lip region end to posterior end of pharyngeal glands (142.5, 130.0–152.0), anal body width(15.8, 14.0–17.5), tail length (20.3, 15.0–25.0), tailannules (11.6, 10.0–14.0), a (25.0, 21.4–27.1), b (5.0, 4.4–5.7),c (35.4, 30.2– 41.7), c9 (1.3, 1.0–1.6), and V (61.8%, 60.0–63.7).The spiral nematode was identi?ed as Helicotylenchus microlobus according to morphologicalandmorphometriccharacteristics(Subbotinetal.,2015).DNAwasextractedfromsinglenematodes(n=8)usingthe Proteinase K method (Kumari and Subbotin, 2012).The internal transcribed spacer (ITS) region of rDNA was ampli?ed with the primers rDNA2/rDNA1.58S (Cherry et al., 1997).The PCR products were then puri?ed and sequenced.The consensus ITS rDNA sequence (accession no.KY271078, 822 bp) that was deposited into the GenBank shared 99% identity with two isolates of H.microlobusfromCalifornia(KM506860.1andKM506859.1)andoneisolateofH.microlobusfromSpain(KM506862.1)(Subbotinetal., 2015).It had only 91% sequence identity with seven isolates of H.pseudorobustus (KM506875.1, KM506880.1, KM506876.1, KM506874.1, KM506872.1, KM506879.1, and KM506878.1) from California, Switzerland, and New Zealand, a spiral nematode species very closely related to H.microlobus in morphology.The molecular tests con?rmed the identity of this spiral nematode as H.microlobus.The H.microlobus nematode was reported as one of the most commonly observed spiral nematodes in soil samples in the stateofMinnesota,andall13soybeancultivarstestedexceptHawkeyewereratedashosts(Taylor,1960).Toourknowledge,thisisthe ?rst report of H.microlobus in North Dakota.