Plant Parasitic Nematodes on Soybean in Expanding Production Areas of Temperate Regions

Journal of Plant Diseases and Protection

https://doi.org/10.1007/s41348-018-0188-y (0123456789().,-volV)(0123456789().,-volV)

ORIGINAL ARTICLE

Plant parasitic nematodes on soybean in expanding production areas of temperate regions

1,2 1 1 Ahmed Elhady • Holger Heuer • Johannes Hallmann

Received: 18 June 2018 / Accepted: 8 September 2018 Ó Deutsche Phytomedizinische Gesellschaft 2018

Abstract Soybean [Glycine max (L.) Merrill] is the most economically important legume in the world. One of the main yield limiting factors is plant parasitic nematodes, such as Heterodera glycines, Meloidogyne incognita or Pratylenchus brachyurus, which cause severe damage in the main production regions of soybean around the world. For Germany, soybean is still a relatively newly cultivated crop and so far, no information about the occurrence and damage potential of plant parasitic nematodes on soybean is available for this region. For a successful growth of soybean, farmers need to understand, which plant parasitic nematodes can be of risk for soybean production. Within this respect, the objective of this study was to describe the spectrum and abundance of plant parasitic nematodes occurring on soybean under temperate conditions in Germany. Data were collected from farmer fields and experimental sites throughout Germany between 2014 and 2015. Representative soil samples were taken before planting and shortly after harvest. Plant parasitic nematodes were extracted by centrifugal floatation, and identified morphologically to genus level. Selected individuals of Pratylenchus were further identified to species level based on COI gene sequences. The population dynamics of Pratylenchus penetrans and co- occurring plant parasitic nematodes on soybean cv. Merlin was studied in microplots. Finally, the impact of P. penetrans affecting nitrogen fixation by the symbiotic bacterium Bradyrhizobium japonicum was studied in greenhouse experiments. Our findings indicated that Pratylenchus is widely spread in soybean fields in Germany. Main species were P. neglectus and P. crenatus and to a lesser extent P. penetrans. In the microplot experiment, nematode multiplication was highest for P. penetrans, followed by Rotylenchus robustus and Paratylenchus projectus. Infection of soybean by P. penetrans significantly reduced nitrogen fixation as indicated by lower numbers of bacteroids and reduced concentration of ureides In conclusion, Pratylenchus spp. are considered to be the most threatening plant parasitic nematodes for soybean production under the temperate conditions of Germany.

Keywords Monitoring Á Occurrence Á Plant parasitic nematodes Á Soybean Á Germany

Introduction providing an environmental sound alternative to synthetic N application. Biological N ﬁxation is of increasing interest Soybean is one of the most important protein sources for in modern crop rotations, both in conventional as well as human consumption and animal feed worldwide. In addi- organic agriculture. Over the last few years, the soybean tion, soybean is equally important for its N ﬁxing capacity production area in Germany has raised from 1000 ha in 2003 to more than 17,000 ha in 2017 and is still growing in the course of a European wide strategy to promote protein & Johannes Hallmann crops. Due to the temperate conditions, cultivars of the [email protected] early maturing ‘‘00’’ and ‘‘000’’ groups are exclusively 1 Department of Epidemiology and Pathogen Diagnostics, grown. Similar to other crops, soybean is exposed to Julius Ku¨hn-Institute, Federal Research Centre for Cultivated numerous abiotic and biotic stresses, of which plant para- Plants (JKI), Toppheideweg 88, sitic nematodes play a major role. 48161 Braunschweig, Mu¨nster, Germany Soybeans are mainly grown in warmer climates where 2 Department of Plant Protection, Faculty of Agriculture, plant parasitic nematodes such as Heterodera glycines, Benha University, Benha, Egypt 123 Journal of Plant Diseases and Protection

Meloidogyne incognita or Pratylenchus brachyurus cause Three weeks before nematode extraction, the soil samples major damage to the crop. Fortunately, those nematode were incubated at 20 °C to stimulate nematode movement species do not occur in Germany. Throughout our soil from the organic into the mineral fraction of the soil. samplings, we observed that other plant parasitic nematode Nematode extraction was then carried out from 100 ml soil species, especially within the genus Pratylenchus, are wide aliquots by centrifugal floatation using magnesium sulfate spread and may become a threat to soybean production in with a specific density of 1.15 (EPPO 2013). Plant parasitic Germany. Pratylenchus species are migratory endopara- nematodes were counted at genus level at 25 9 magnifi- sites that move through the root cortex. As a result of cation using a light microscope, and the Pi and Pf values nematode movement and plant defense reaction, root tissue were used to calculate the multiplication rate (Pf/Pi = final is destroyed and lesions are formed that become visible as population density/initial population density). dark brown rotten spots. Species of Pratylenchus are cos- mopolitan and in most cases have a very wide host range Molecular identification of Pratylenchus species covering monocotyledon and dicotyledonous crops (Smiley 2010). The life cycle of Pratylenchus species varies For species identification of Pratylenchus, individual between 3 and 9 weeks according to species, host plant and nematodes were hand-picked and transferred to 1.5-ml environmental conditions (Jones and Fosu-Nyarko 2014). microtubes for nematode lysis and DNA extraction. Besides causing direct damage to the plant, Praty- Briefly, an equal volume of 10 ll lysis buffer was added to lenchus can disturb N fixation by interfering with the tube. The lysis buffer contained 0.2 M NaCl, 0.2 M Bradyrhizobium japonicum, the symbiotic N fixing rhi- Tris–HCl (pH 8.0), 1% (v/v) b-mercaptoethanol, and zobacterium of soybean. B. japonicum is a beneficial 800 lg/ml proteinase-K. The microtubes were then incu- microbe that interacts with its host via chemical and bated in a thermomixer (Eppendorf, Hamburg, Germany) molecular dialog. In principle, soybean produces flavo- at 65 °C and 750 rpm for 2 h, followed by 5 min incuba- noids which stimulate the rhizobium to form nodules in tion at 100 °C, and stored at - 20 °C. The ribosomal 18S order to deliver fixed nitrogen to the plant (Badri et al. rRNA was amplified using the primers modified from 2009). If Pratylenchus competes in soybean roots with B. Blaxter et al. (1998): SSU18A ? 250-TCAAA- japonicum for plant resources this may lead to reduced N GATTAAGCCATGCATG-30 and Nem1078r 50- fixation and the overall positive pre-crop effect of soybean CCGGAAGACTTTCGTTTCC-30. The mitochondrial within rotations is reduced or even lost. cytochrome oxidase 1 gene (CO1) was amplified by primer Thus, the objectives of this study were (1) to evaluate JB3 50-TTTTTTGGGCATCCTGAGGTTTAT-30 (Bowles the taxa of plant parasitic species and in particular the et al. 1992) and primer JB5 50-AGCACCTAAACT- species of Pratylenchus associated with soybean produc- TAAAACATAATGAAAATG-30 (Derycke et al. 2008). tion in Germany, (2) to investigate the population dynamics The PCR products were purified with GeneJET PCR of plant parasitic nematodes on different soybean cultivars, Purification Kit (Thermo Fisher Scientific, Schwerte, Ger- and (3) to analyze the impact of Pratylenchus penetrans on many). The purified products were sent to Macrogen, the nitrogen fixation of soybean. Netherlands, for Sanger sequencing in both directions. A CO1 database for Pratylenchus was generated based on 18S rRNA sequences of known Pratylenchus species pro- Materials and methods vided by Dr. Lieven Waeyenberge, ILVO, Belgium. DNA sequences were deposited at NCBI GenBank under Soil sampling, microscopic identification accession numbers MH394226–MH394245. and quantification of plant parasitic nematodes associated with soybean Population dynamics of plant parasitic nematodes on soybean To describe the occurrence and abundance of plant parasitic nematodes in soybean, soil samples were collected The population dynamics of plant parasitic nematodes on from 27 soybean sites throughout Germany over three soybean were studied in 1 m2 microplots at the JKI successive years (Table 1). In general, samples were taken experimental site in Mu¨nster, Germany. In 2014, twenty in May immediately before soybean planting to record the microplots naturally infested with plant parasitic nema- initial population density (Pi) and in September to October todes were planted with soybean cv. Merlin. Soil samples after harvest of the soybean to detect the final population for Pi were taken on April 3, 2014, and for Pf on October (Pf). Each sample consisted of 30 cores (0–30 cm) taken in 20, 2014. Each sample consisted of 30 cores from the top a zigzag pattern from the entire field. The collected soil 20 cm of soil. The soil was sieved at 8 mm, mixed, and an was mixed and stored at 4 °C until further processing. aliquot of 250 ml soil was extracted for nematodes by 123 Journal of Plant Diseases and Protection

Table 1 The regions, cultivars Samples no. Sampling code Region Cultivar Year and years of soybean ﬁeld samplings 1 S_M_14 Seewiesenhof, Augsburg, Mu¨nchen Merlin 2014 2 B_P_14 Biedesheim, Mannheim Primus 2014 3 B_M_14 Biedesheim, Mannheim Merlin 2014 4 B_S_14 Biedesheim, Mannheim Sultana 2014 5 H_P_14 Herxheim, Rheinland-Pfalz Primus 2014 6 H_M_14 Herxheim, Rheinland-Pfalz Merlin 2014 7 H_S_14 Herxheim, Rheinland-Pfalz Sultana 2014 8 S_M_14 Aschau am Inn, Waldkraiburg, Mu¨nchen Merlin 2014 9 S_S_14 Aschau am Inn, Waldkraiburg, Mu¨nchen Sultana 2014 10 B_P_15 Biedesheim, Mannheim Primus 2015 11 B_M_15 Biedesheim, Mannheim Merlin 2015 12 B_S_15 Biedesheim, Mannheim Sultana 2015 13 RN_U_15 Nagold-Vollmaringen near Tu¨bingen Unknown 2015 14 H_P_15 Rheinstetten, Ettlingen, Karlsruhe Primus 2015 15 P_P_15 Rheinstetten, Ettlingen, Karlsruhe Primus 2015 16 R_P_15 Rheinstetten, Ettlingen, Karlsruhe Primus 2015 17 H_P_15 Herxheim, Rheinland-Pfalz Primus 2015 18 H_M_15 Herxheim, Rheinland-Pfalz Merlin 2015 19 H_S_15 Herxheim, Rheinland-Pfalz Sultana 2015 20 OF_P_15 Ober Flo¨rsheim, Mannheim Primus 2015 21 OF_M_15 Ober Flo¨rsheim, Mannheim Merlin 2015 22 OF_S_15 Ober Flo¨rsheim, Mannheim Sultana 2015 23 LFG_U_15 Umkirch, Freiburg im Breisgau Unknown 2015 24 NLFLB_M_15 Niederhummel, Langenbach, Mu¨nchen Merlin 2015 25 NLFLB_S_15 Niederhummel, Langenbach, Mu¨nchen Sultana 2015 26 NH_U15 Hohnhorst, Hannover, Niedersachsen Unknown 2015 27 Z_S_15 Zalf, Mu¨ncheberg Sultana 2015 centrifugal ﬂotation as described above. One day before contaminants. After 3 days, the B. japonicum culture was planting, each plot received 60 g complete fertilizer (12% centrifuged at 4000 g for 10 min. The pellet was washed N, 12% K, 12% P, 2% Mg, 11% S, RIGK, Wiesbaden, twice with sterile tap water to remove antibiotic residues

Germany). Soybean was planted on May 21, 2014, at 70 and finally adjusted to OD600 = 0.2 by dilution with sterile seeds/m2 representing 100 kg/ha. Seeds were equally dis- tap water. tributed over three rows of 1 m length and 30 cm distance between rows. Plots were watered as necessary and plants Greenhouse experiment were harvested on October 20, 2014. In order to investigate whether nitrogen fixation by B. Effect of P. penetrans on N2 fixation by B. japonicum in soybean is affected by P. penetrans,we japonicum in soybean designed an experiment with two treatments. Both treatments were inoculated with B. japonicum, however, only B. japonicum growth conditions one treatment was treated at the same time with P. penetrans. Briefly, soybean seeds cv. Primus were first surface B. japonicum was isolated from Histick Soy (BASF, Lud- sterilized with 1.5% sodium hypochlorite for 15 min to wigshafen, Germany), and a rifampicin resistant mutant exclude microbial contamination and then grown for was selected from a high-density culture. Inoculum of B. 5 days at room temperature on moist paper tissue. Seed- japonicum was produced by growing the bacterium in lings of similar growth were selected and transferred into Yeast Mannitol (YM) liquid medium at 28 °C. The med- pots containing 500 ml of a field soil/sand mixture (1:2, ium was supplemented with 1 mg l-1 of vancomycin and v:v). The chemical analysis of soil used in the study is 50 mg l-1 of rifampicin to suppress microbial described in supplementary Table 1. After 1 week, soybean

123 Journal of Plant Diseases and Protection seedlings were inoculated with 4 ml of a B. japonicum cell Results suspension (OD600 = 0.2) and 4 ml nematode suspension containing 1000 mixed stages of P. penetrans. Pots were Occurrence and abundance of plant parasitic kept in the greenhouse at 24 °C and 16 h photoperiod and nematodes associated with soybean in Germany watered every 2 days. A total of seven genera of plant parasitic nematodes were Soybean sampling found to be associated with soybean production (Table 2). The occurrence and density levels of plant parasitic One month after inoculation, plants were sampled and nematodes before and after soybean cultivation showed evaluated for N2 fixation and P. penetrans invasion. Nod- that the most dominant genus associated with soybean as a ule numbers and biomass per plant were measured. Next, host was Pratylenchus. It was present in all soil samples 0.05 g nodule biomass representing 3–7 nodules was collected and reached population densities of up to 1380 selected and surface sterilized for 10 min in 1% sodium nematodes/100 ml soil. Other commonly detected taxa hypochlorite. Nodules were rinsed with sterilized water, were Helicotylenchus, Paratylenchus and Tylen- transferred to 2 ml microtubes and squeezed with the tip of chorhynchus. Less frequently found genera included Am- a plastic pipette. Tubes were filled up with 1 ml sterile plimerlinius, Ditylenchus and Meloidogyne. Heterodera 10 mM MgCl2 and homogenized by vortexing for 15 s. and Trophurus occurred in one sample each, and due to Serial dilutions were plated onto YM Agar supplemented their low distribution are not included in Table 2. The with 1 mg l-1 of vancomycin and 50 mg l-1 of rifampicin, average multiplication rate of Pratylenchus on soybean and incubated at 28 °C. After 3 days, colony-forming units over all fields was 3 and varied between 0.6 (Flo¨rsheim, (CFU) of bacteroids, representing the nitrogen-fixing form Merlin) and 10 (Biedesheim, Merlin). Among other factors, that converts atmospheric nitrogen into ammonia, were multiplication rate of Pratylenchus was affected by the counted. soybean cultivar. As shown for Biedesheim in 2014, the multiplication rate of Pratylenchus was higher on the Quantification of ureides soybean cultivars Primus and Merlin than on the cultivar Sultana (Table 2). Other than Pratylenchus, nematode To measure the nitrogen-fixing capacity, 0.2 g aliquots of multiplication was observed for Helicotylenchus and freeze-dried shoot tissue were prepared. Thereafter, 200 ll Paratylenchus on most of the tested sites. For Tylen- of ice-cold sterile deionized H2O water was added and the chorhynchus, results varied between a slight increase in plant tissue ground with a plastic micropestle. Plant tissue population size and a reduction in population size. Popu- adhering to the micropestle was carefully rinsed off with lation densities of Amplimerlinius, Meloidogyne and Dity- 800 ll sterile H2O. The suspension was then passed lenchus declined following soybean cultivation (Table 2). through one layer of Miracloth (Merck, Darmstadt, Ger- many). The filtrate was transferred to 1.5-ml microtubes Predominant species of Pratylenchus associated and centrifuged at 20,000 g at 4 °C for 30 min. Two with soybean cultivation in Germany microtubes were each filled with 100 ll of the filtrate, one used for measuring total ureides and the other for mea- The CO1 gene sequences of individual Pratylenchus suring allantoic acid based on a photometric assay using specimen picked randomly from each soybean field at phenylhydrazine and potassium ferricyanide (Collier and harvest time identified P. neglectus and P. crenatus as the Tegeder 2012). The allantoin concentration was calculated most abundant species of Pratylenchus associated with by subtracting allantoic acid from total ureides. A 10 mM soybean (Table 3). P. penetrans was only detected in stock solution of allantoin C 98.0% (Sigma-Aldrich, ZALF soybean field with a Pf/Pi of 2.74. The identity of Munich, Germany) was used to prepare a standard curve by the COI sequences ranged between 96 and 100% when serial dilution. All reactions were measured within 30 min blasted against the CO1 sequences of different Praty- of mixing at 520 nm absorbance using a lenchus species that have been identified based on their 18S spectrophotometer. rRNA sequences and morphology. Interestingly, at Bun- desallee in Braunschweig, recovered Pratylenchus from the soil were confirmed as P. neglectus, whereas those recovered from roots were identified as P. crenatus.

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Table 2 Plant parasitic nematodes before planting (Pi) and after harvest (Pf) of soybean in production areas of Germany Sampling code (see Pratylenchus Helicotylenchus Paratylenchus Tylenchorhynchus Table 1) Pi/ Pf/ Pf/Pi Pi/ Pf/ Pf/ Pi/ Pf/ Pf/ Pi/ Pf/ Pf/ 100 ml 100 ml 100 ml 100 ml Pi 100 ml 100 ml Pi 100 ml 100 ml Pi soil soil soil soil soil soil soil soil

S_M_14 64 183 2.85 250 469 1.87 0 0 0 0 0 0 B_P_14 80 744 9.35 216 516 2.39 552 816 1.48 24 60 2.50 B_M_14 64 664 10.3 224 584 2.60 432 768 1.78 24 88 3.66 B_S_14 112 468 4.17 304 528 1.73 408 528 1.30 8 84 10.5 H_P_14 104 458 4.40 0 0 0 228 286 1.25 64 227 3.54 H_M_14 120 140 1.16 0 0 0 208 273 1.31 30 170 5.66 H_S_14 92 260 2.82 0 0 0 244 317 1.30 30 218 7.26 S_M_14 58 85 1.45 124 66 0.53 0 0 0 0 0 0 S_S_14 36 89 2.47 92 52 0.56 0 0 0 0 0 0 B_P_15 86 108 1.26 0 0 0 96 107 1.11 0 0 0 B_M_15 85 81 0.95 0 0 0 137 79 0.57 0 0 0 B_S_15 88 75 0.85 0 0 0 187 90 0.48 0 0 0 RN_U_15 856 1620 1.89 56 276 4.92 8 0 0 24 120 5.00 H_P_15 184 1100 5.97 16 0 0 0 0 0 8 128 16.0 P_P_15 432 376 0.87 280 20 0.07 24 4 0.16 8 20 2.50 R_P_15 176 1380 7.84 24 12 0.50 0 0 0 72 0 0 H_P_15 86 99 1.15 0 0 0 1716 694 0.40 21 0 0 H_M_15 70 147 2.10 0 0 0 2156 4605 2.13 0 0 0 H_S_15 108 72.5 0.67 0 0 0 1526 3283 2.15 0 0 0 OF_P_15 26 45 1.73 14 8 0.57 16 96 6.00 0 0 0 OF_M_15 52 29 0.55 8 7 0.87 48 90 1.87 12 0 0 OF_S_15 121 154 1.27 10 7 0.70 66 129 1.95 8 0 0 LFG_U_15 20 90 4.52 0 0 0 4 90 22.5 0 0 0 NLFLB_M_15 108 144 1.33 0 0 0 0 0 0 16 28 1.75 NLFLB_S_15 40 276 6.96 12 24 2.00 0 0 0 8 20 2.50 NH_U15 788 784 0.99 0 0 0 a 20 a 64 56 0.87 Z_S_15 372 1020 2.74 0 0 0 56 140 2.50 88 12 0.13 Sampling code (see Amplimerlinius Ditylenchus Meloidogyne J2 Non plant parasitic Table 1) nematodes Pi/100 ml Pf/100 ml Pf/ Pi/100 ml Pf/100 ml Pf/ Pi/100 ml Pf/100 ml Pf/ Pi/100 ml Pf/100 ml soil soil Pi soil soil Pi soil soil Pi soil soil

S_M_14 0 0 0 0 0 0 0 0 0 554 2729 B_P_14 0 0 0 0 0 0 0 0 0 1584 1956 B_M_14 0 0 0 0 0 0 0 0 0 1000 6776 B_S_14 0 0 0 0 0 0 0 0 0 1344 2520 H_P_14 0 0 0 0 0 0 294 0 0 1354 1741 H_M_14 0 0 0 0 0 0 196 0 0 1482 1052 H_S_14 0 0 0 0 0 0 160 0 0 1500 1219 S_M_14 0 0 0 0 0 0 0 0 0 1060 426 S_S_14 0 0 0 0 0 0 0 0 0 1168 616 B_P_15 0 0 0 60 0 0 0 0 0 3548 551 B_M_15 0 0 0 34 0 0 0 0 0 1517 746 B_S_15 0 0 0 43 0 0 0 0 0 1451 998 RN_U_15 112 0 0 0 0 0 0 0 0 5304 1236 H_P_15 392 0 0 0 0 0 0 0 0 3536 2450

123 Journal of Plant Diseases and Protection

Table 2 (continued) Sampling code (see Amplimerlinius Ditylenchus Meloidogyne J2 Non plant parasitic Table 1) nematodes Pi/100 ml Pf/100 ml Pf/ Pi/100 ml Pf/100 ml Pf/ Pi/100 ml Pf/100 ml Pf/ Pi/100 ml Pf/100 ml soil soil Pi soil soil Pi soil soil Pi soil soil

P_P_15 8 0 0 0 0 0 0 0 0 848 2388 R_P_15 8 0 0 0 0 0 0 0 0 896 3468 H_P_15 0 0 0 0 0 0 72 0 0 2892 5361 H_M_15 0 0 0 0 0 0 34 0 0 2404 982 H_S_15 0 0 0 0 0 0 46 0 0 1574 1145 OF_P_15 0 0 0 0 0 0 0 0 0 1908 989 OF_M_15 0 0 0 0 0 0 0 0 0 1726 1181 OF_S_15 0 0 0 0 0 0 0 0 0 2415 1269 LFG_U_15 0 0 0 0 0 0 0 0 0 2036 3498 NLFLB_M_15 0 0 0 0 0 0 0 0 0 1120 0 NLFLB_S_15 0 0 0 0 0 0 0 0 0 1620 624 NH_U15 0 0 0 0 0 0 0 0 0 328 0 Z_S_15 0 0 0 0 0 0 0 0 0 1396 620 aMissing Pi; Pf/Pi could not be calculated

Table 3 Identiﬁcation of the dominant species of Pratylenchus associated with soybean in Germany, based on cytochrome oxidase 1 (CO1) sequencing Isolation source Pratylenchus species Identity based on CO1 (%)

Soybean roots, cv. Primus, Bundesallee region, 2017 P. crenatus 99 Soybean soil after harvest, cv. Primus, Bundesallee region, 2017 P. neglectus 96 Soybean soil after harvest, cv. Primus; Merlin and Sultana, Herxheim region, 2015 P. crenatus 99 Soybean soil after harvest, cv. Primus; Merlin and Sultana, Herxheim region, 2015 P. neglectus 97 Soybean soil after harvest, cv. Primus; Merlin and Sultana, Groß Lu¨sewitz region, 2016 P. neglectus 97 Soybean soil after harvest, cv. Unknown, Nagold-Vollmaringen region, 2015 P. neglectus 99 Soybean soil after harvest, cv. Primus Rheinstetten region, 2016 P. neglectus 99 Soybean soil after harvest, cv. Primus, Rheinstetten region, 2015 P. neglectus 97 Soybean soil after harvest, cv. Primus, Rheinstetten region, 2016 P. neglectus 86 Soybean soil after harvest, cv. Sultana, Zalf, 2015 P. penetrans 99 Soybean soil after harvest, cv. Primus; Merlin and Sultana, Biedesheim region, 2015 P. neglectus 100 The percentages (%) indicate the similarity of mitochondrial cytochrome oxidase 1 gene (CO1) of Pratylenchus species as compared to NCBI and local databases

Population dynamics of plant parasitic penetrans with 2442 nematodes per 100 ml soil, which was nematodes on soybean significantly higher than for R. robustus (228 nematodes/ 100 ml soil) and P. projectus (130 nematodes/100 ml soil). The microplots were naturally infested with P. penetrans, Similarly, the multiplication rate was highest for P. pene- Rotylenchus robustus and Paratylenchus projectus trans (Pf/Pi = 66.6), which was significantly higher than (Table 4). All three plant parasitic nematode species were for R. robustus (7.4) and P. projectus (2.7). Visual able to reproduce on soybean cv. Merlin (Figs. 1, 2). The inspection of the soybean roots at harvest indicated that P. highest final population density was observed for P. penetrans has colonized the entire root system (Fig. 3).

123 Journal of Plant Diseases and Protection

Table 4 Plant parasitic nematodes in microplots before planting (Pi) and after harvest (Pf) of soybean Microplot Pratylenchus Paratylenchus Rotylenchus Pi/100 ml soil Pf/100 ml soil Pf/Pi Pi/100 ml soil Pf/100 ml soil Pf/Pi Pi/100 ml soil Pf/100 ml soil Pf/Pi

16 60 944 16 0 0 356 1008 3 17 28 1008 36 0 0 164 528 3 18 80 780 10 0 0 48 564 12 19 152 1740 11 8 0 0 24 228 10 20 28 420 15 0 0 164 1188 7 21 0 1176 0 0 0 12 22 96 600 6 96 252 3 80 792 10 23 20 960 48 4 0 0 32 240 8 46 64 2604 41 32 108 3 0 0 47 116 2472 21 208 804 4 0 0 48 36 4632 129 144 168 1 0 0 49 48 4092 85 68 84 1 0 0 50 20 3372 169 16 48 3 0 0 51 108 4092 38 292 360 1 0 0 52 84 2472 29 76 96 1 0 0 53 20 3576 179 96 264 3 0 0 54 20 1452 73 24 36 2 0 0 55 20 5940 297 16 252 16 0 0 56 96 3384 35 32 96 3 0 0 57 108 3120 29 72 36 1 0 0

Fig. 1 Soybean cultivar Merlin grown in microplots to study their host status for different plant parasitic nematodes

Effect of P. penetrans on N2 fixation by B. japonicum in soybean

Root nodulation on soybean cv. Primus was severely Fig. 2 Abundance of different plant parasitic nematodes before and affected by P. penetrans. Nodule numbers per plant were after cultivation of soybean cv. Merlin in microplots at Mu¨nster, significantly reduced in the presence of P. penetrans Germany. Mean ± SD (n = 20 microplots), Pf/Pi was calculated (19 ± 10) compared to the non-inoculated control based on the log transformed number of nematodes. The Pi values (37 ± 15). Similarly, nodule biomass was significantly indicate the initial population density prior to soybean cultivation; Pf value indicate the final population density after soybean harvest, and lower in the presence of P. penetrans. Numbers of Pf/Pi values indicate the multiplication rate

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Discussion

A better awareness of the most abundant plant parasitic nematodes on soybean and their potential impact on soybean production will decide about the need to develop control strategies. This study described the genera of plant parasitic nematodes associated with soybean grown under temperate conditions in Germany. The dominant genus throughout all field sites was Pratylenchus. The main species within this genus were P. neglectus and P. crenatus, and to a lesser extent P. penetrans. All three Praty- lenchus species occur worldwide and have previously been reported from soybean (Ferris and Bernard 1967; Fourie Fig. 3 Soybean roots from Bundesallee field colonized by Praty- et al. 2001). Pratylenchus species are among the most lenchus sp., stained with acid fuchsin dominant plant parasitic nematode taxa in Germany (Hallmann et al. 2007). They can easily multiply on crops rotated with soybean such as cereals and maize and thus impose the risk to build up to damaging levels. Quite interestingly, P. crenatus was detected in soybean roots while at the same time P. neglectus was more abundant in soil. This could indicate that P. crenatus may outcompete P. neglectus on soybean as a host. It was demonstrated that in mixed populations of plant parasitic nematodes, typi- cally one of the species reproduces much better than the others on a given host plant (Eisenback 1993). In our microplot experiment, the high multiplication rate of P. penetrans compared to Rotylenchus robustus and Paratylenchus projectus reflected the high susceptibility of soybean as a host crop for P. penetrans. Plant parasitic nematodes differ in their multiplication rates among host Fig. 4 Pratylenchus penetrans Effect of on total ureides (allantoin plants and even genotypes (Castillo and Vovlas 2007; and allantoate), the forms of fixed N2 by Bradyrhizobium japonicum– soybean symbiosis. The asterisk indicates significant differences Smiley et al. 2013). Our findings are in line with the results between the treatments at P \ 0.05 (*) and P \ 0.01 (**) of Be´lair et al. (2002) who found that P. penetrans multi- plies very efficient on soybean. B. japonicum in nodules and the concentration of ureides, In our study, N2 fixation of soybean by B. japonicum i.e., the transport form of fixed nitrogen, were significantly was affected by P. penetrans. The number and biomass of lower in soybean infested with P. penetrans than in the nodules were significantly reduced in soybean plants control (Fig. 4). Shoot and root fresh weight and shoot dry infected with P. penetrans compared to uninfested con- weight were not significantly affected by the presence of P. trols. Furthermore, the viability of bacteroids and the penetrans (Table 5). amount of fixed nitrogen (allantoic acid and allantoin) was significantly lower in nematode-infected plants. The fact that plant parasitic nematodes interfere with root

Table 5 Effect of Pratylenchus penetrans on soybean plant growth and root nodulation by Bradyrhizobium japonicum Treatments Shoot fresh Root fresh Shoot dry Nodules per Nodule weight Bacteroids (107 per weight (g) weight (g) weight (g) plant per plant (g) 0.05 g nodules)

B. japonicum 3.2 ± 0.6a 2.3 ± 0.39a 0.7 ± 0.10a 37.2 ± 14.6a 0.2 ± 0.03a 8.7a B. japonicum ? P. penetrans 3.0 ± 0.05a 1.9 ± 0.36a 0.56 ± 0.08a 19.1 ± 10.3b 0.07 ± 0.05b 1.3b Mean ± standard deviation with different letters within a column indicate signiﬁcant differences according to Tukey’s test (P \0.05, n =10)

123 Journal of Plant Diseases and Protection nodulation and nitrogen fixation was already observed for References other nematode–legume interactions. For instance, soybean infected with Pratylenchus pseudorobustus showed fewer Badri D, Weir T, Daniel L, Vivanco J (2009) Rhizosphere chemical nodules and less N fixation (Germani et al. 1984). Simi- dialogues: plant–microbe interactions. Curr Opin Biotechnol 2 20:642–650. https://doi.org/10.1016/j.copbio.2009.09.014 larly, peas infected with H. goetingiana showed fewer Ballhorn DJ, Younginger BS, Kautz S (2014) An aboveground nodules (Jones and Moriarty, 1956). In general, it is pathogen inhibits belowground rhizobia and arbuscular mycor- assumed that P. penetrans invasion and migration in the rhizal fungi in Phaseolus vulgaris. BMC Plant Biol 14:321. root will destroy the root tissue and thus reduce the https://doi.org/10.1186/s12870-014-0321-4 Be´lair G, Fournier Y, Dauphinais N, Dangi OP (2002) Reproduction transportation of fixed nitrogen to the shoot. Also, it was of Pratylenchus penetrans on various rotation crops in Quebec. shown that the defense response of plants to pathogens can Phytoprotection 83:111–114. https://doi.org/10.7202/706233ar affect the symbiotic relationship and N2 fixation (Faessel Blaxter ML, de Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete et al. 2010; Ballhorn et al. 2014). A, Vanfleteren JR, Mackey LY, Dorris M, Frisse LM, Vida JT, Thomas WK (1998) A molecular evolutionary framework for the In summary, this study has identified the main plant phylum Nematoda. Nature 392:71–75. https://doi.org/10.1038/ parasitic nematodes associated with soybean grown under 32160 temperate conditions in Germany. Within a microplot Bowles J, Blair D, Mcmanus D (1992) Genetic variants within the experiment, the good host status of soybean enabling P. genus Echinococcus identified by mitochondrial DNA sequencing. Mol Biochem Parasitol 54:165–173. https://doi.org/10.1016/ penetrans multiplication of up to 66 times was confirmed. 0166-6851(92)90109-W Unfortunately, the damage potential of P. penetrans on Castillo P, Vovlas N (2007) Pratylenchus, (Nematoda, Pratylenchi- soybean in the field was not covered in this study. How- dae): diagnosis, biology, pathogenicity and management (nema- ever, as shown in pot experiments, P. penetrans infestation tology monographs and perspectives). Nematology 6:529 Collier R, Tegeder M (2012) Soybean ureide transporters play a reduced overall N2 fixation of the soybean crop thus min- critical role in nodule development, function and nitrogen imizing its contribution to increase soil nitrogen. The study export. Plant J 72:355–367. https://doi.org/10.1111/j.1365-313X. will increase our awareness for plant parasitic nematodes 2012.05086.x as potential pathogens of soybean grown in temperate Derycke S, Remerie T, Backeljau T, Vierstraete A, Vanfleteren J, Vincx M, Moens T (2008) Phylogeography of the Rhabditis regions and their negative impact on nitrogen fixation. This (Pellioditis) marina species complex: evidence for long-distance might provide the ground to develop new strategies for dispersal, and for range expansions and restricted gene flow in improvement of N fixation under nematode pressure by the northeast Atlantic. Mol Ecol 17:3306–3322. https://doi.org/ proper nematode management. 10.1111/j.1365-294X.2008.03846.x Eisenback JD (1993) Interactions between nematodes in cohabitance. In: Khan MW (ed) Nematode interactions. Springer, Dordrecht, Acknowledgements We thank Dr. Kristina Bachteler, Agricultural pp 134–174 Centre for Soybean Cultivation and Development, Taifun-Tofu EPPO (2013) European and mediterranean plant protection organi- GmbH, for providing soybean seeds. We also thank Prof. Dr. zation: PM 7/119 (1) nematode extraction. EPPO Bull Mechthild Tegeder, School of Biological Sciences, Washington State 43:471–495. https://doi.org/10.1111/epp.12077 University, for sharing the protocol for determining total ureides. Mrs. Faessel L, Nassr N, Lebeau T, Walter B (2010) Chemically-induced Elvira Woldt and Mrs. Agnes Wind are acknowledged for their resistance on soybean inhibits nodulation and mycorrhization. excellent technical assistance. Plant Soil 329:259–268. https://doi.org/10.1007/s11104-009- 0150-7 Funding Elhady was funded by the German Egyptian Research Long- Ferris VR, Bernard RL (1967) Population dynamics of nematodes in term Scholarship GERLS 57076387, and his participation in confer- fields planted to soybeans and crops grown in rotation with ences was funded by the Gisela und Hermann Stegemann Foundation soybeans. I. The genus Pratylenchus (Nemata: Tylenchida). and the Gemeinschaft der Fo¨rderer und Freunde des Julius Ku¨hn- J Econ Entomol 60:405–410. https://doi.org/10.1093/jee/60.2. Instituts. 405 Fourie H, McDonald A, Loots G (2001) Plant-parasitic nematodes in Compliance with Ethical Standards field crops in South Africa. 6. Soybean Nematol 3:447–454. https://doi.org/10.1163/156854101753250773 Germani G, Mugnier J, Dommergues Y (1984) Influence of Conflict of interest The authors declare that they have no conflict of pathogenic nematodes on nodulation and seed yield of soybeans interest. in Senegal. Revue Nematol 7:335–340 Hallmann J, Frankenberg A, Paffrath A, Schmidt H (2007) Occur- Ethical standards The manuscript has not been submitted to any other rence and importance of plant-parasitic nematodes in organic journal and has not published previously (partly or in full). A single farming in Germany. Nematology 9:869–879. https://doi.org/10. study was not split up into several parts to increase the quantity of 1163/156854107782331261 submissions and submitted to various journals or to one journal over Jones MG, Fosu-Nyarko J (2014) Molecular biology of root lesion time. No data have been fabricated or manipulated (including images) nematodes (Pratylenchus spp.) and their interaction with host to support our conclusions. No data, text or theories by others are plants. Ann Appl Biol 164:163–181. https://doi.org/10.1111/aab. presented. 12105 Jones FGW, Moriarty F (1956) Further observations on the effects of peas, beans and vetch upon soil population levels of pea root

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eelworm, Heterodera go¨ttingiana Liebscher. Nematologica Smiley RW, Machado S, Gourlie JA, Pritchett LC, Yan G, Jacobsen 1:268–273. https://doi.org/10.1163/187529256X00104 EE (2013) Effects of crop rotations and tillage on Pratylenchus Smiley RW (2010) Root-lesion nematodes: Biology and management spp. in the semiarid Pacific Northwest United States. Plant Dis in Pacific Northwest wheat cropping systems. A Pacific North- 97:537–546. https://doi.org/10.1094/PDIS-08-12-0788-RE west Extension Publication, Oregon

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