Populations of parasitic nematodes colonizing Jerusalem artichoke (Helianthus tuberosus L.)

The populations of parasitic nematodes colonizing the rhizosphere of Helianthus tuberosus L. grown in Polish plantations were studied in the summer of 2016 and the spring of 2017. A total of 35 nematode species were identified: Paratrichodorus pachydermus, Trichodorus cylindricus, T. viruliferus, Longidorus attenuatus, L. elongatus, L. leptocephalus, Criconema annuliferum, Criconemoides informis, Mesocriconema rusticum, M. solivagum, M. xenoplax, Paratylenchus nanus, P. neoamblycephalus, P. projectus, Bitylenchus dubius, B. maximus, Merlinius brevidens, M. nothus, Scutylenchus quadrifer, S. tartuensis, Helicotylenchus digonicus, H. pseudorobustus, H. vulgaris, Rotylenchus pumilus, R. robustus, Pratylenchus crenatus, P. fallax, P. neglectus, Hirschmanniella gracilis, Aphelenchoides fragariae, Aphelenchus avenae, A. eremitus, Ditylenchus dipsaci, and D. medicaginis. Aphelenchoides fragariae and Ditylenchus dipsaci could be foliar pathogens of H. tuberosus L. This is the first study to demonstrate the presence of A. fragariae on the leaves of the Jerusalem artichoke in Poland. The frequencies of occurrence and population densities of the 35 nematode species were determined.


Introduction
Jerusalem artichoke (Helianthus tuberosus L.) is a versatile plant native to North America, and has a variety of applications, including in energy generation, feed and food production, and phytoreclamation.This species is mostly prized for its tubers and aerial parts.The stems and leaves of Jerusalem artichoke are used as animal feed.The Jerusalem artichoke is an alternative crop for human consumption and has numerous benefits, although it is not very popular in Poland.Its tubers, shoots, and flowers contain valuable phytochemicals that can be used for producing medicines, cosmetics, and food.Jerusalem artichoke is a rich source of inulin and other fructooligosaccharides suitable for the production of functional foods.Inulin induces a smaller increase in blood sugar levels than other carbohydrates do, and it is particularly recommended for diabetic patients [1,2].Inulin has also been found to have probiotic effects in humans [3,4].Jerusalem artichoke tubers are characterized by high content of fructans, dietary fiber, and phenolic compounds.They also contain polyhydric alcohols, peptides, proteins, vitamins, choline, lecithin, polyunsaturated fatty acids, and phytochemicals [5][6][7].The plant is rich in proteins with high biological value, and it contains all exogenous amino acids, including methionine [8].The tubers typically contain 80% water, 15% carbohydrates, and 1-2% proteins, and are also a source of minerals, mostly iron (0.4-3.7 mg 100 g −1 ), calcium (14-37 mg 100 g −1 ), and potassium (420-657 mg 100 g −1 ) [7,8].
Similar to Miscanthus ×giganteus, H. tuberosus L. is particularly suitable for biofuel production.The Jerusalem artichoke is an excellent source of biomass owing to its high yields and high lignin and cellulose content.However, long-term monocultures can compromise the plant's genetic diversity.The plant is an attractive substrate for energy generation, including direct combustion, combined combustion with coal, and biogas production [9,10].
Pathogenic nematodes are small (approximately 1 mm in length, with only Longidorus and Xiphinema reaching up to 6 mm in length) worm-like animals that live in soil and feed on plant roots and stems.Nematodes pierce plant cells with a needle-like structure known as a stylet and suck plant sap.Parasitic nematodes feed on the roots and stems of many plant species in Poland, including the Jerusalem artichoke.Communities of plant and soil nematodes are influenced by various factors, including the soil environment.The plant's health largely determines tolerance to pathogens and parasites.Soil quality is influenced by the structure of the soil food web.Nematodes feed on different types of organic matter and they belong to various trophic types: microbivores that feed on microorganisms; parasites that feed on fungi, algae, and lichens; higher-plant feeders; omnivores; and predators.They are involved in soil processes such as decomposition and circulation of soil organic matter, energy flow, and circulation of plant nutrients [11][12][13].
Herbivorous nematodes and pathogenic bacteria and fungi cause diseases with complex etiologies.Nematodes of the genera Longidorus, Xiphinema, and Trichodorus are vectors of soil-borne viruses that are transmitted to plant roots [14].There is a general scarcity of research into parasitic plant nematodes [15], and nematodes of the genera Longidorus and Xiphinema that colonize H. tuberosus L. in Poland have not been studied so far.Jerusalem artichoke tubers seem to be fairly resistant to nematodes [16].However, long-term monocultures of energy crops can compromise plant resistance to pathogens and diseases.In view of the above, the objective of this study was to analyze the populations of parasitic nematodes colonizing the root zone of H. tuberosus L. in Poland.

Material and methods
The study was carried out in the summer of 2016  Province.
The soil of the areas where the trials were conducted was heavy loam.The agronomic soil category was heavy soil, with the overall sum of fractions less than 0.02 mm amounting to 37.7%.Based on particle size, the soil can be categorized as heavy soil, with its agricultural suitability classified as class IV according to the Polish soil classification system.
According to the data from the Institute of Meteorology and Water Management in Warsaw, mean total precipitation in 2016 was lower (455 mm) than the multiannual mean (610 mm) precipitation, while in 2017, it was higher (755 mm).The rainfall observed in July in 2016 and 2017 beneficially affected the level of moisture on the soil surface, as well as the growth and development of root crops.During the remaining months, precipitation was significantly lower.Mean air temperature during the first vegetation season (2016), 11.3°C, was similar to mean air temperature during the second vegetation season (11.4°C); both values were slightly higher than the multiannual mean (10.6°C).During the periods of April-May and June-August, the mean monthly air temperatures were similar to the multiannual mean values.The temperatures observed from October to December were higher than the multiannual mean, by 2°C on average.In 2017, the mean air temperature of 11.3°C exceeded the multiannual mean by 0.7°C.The high temperature in November supported continued vegetation.The weather conditions in December did not pose any significant hazards for the wintering plants.The temperature was favorable for the continued growth of the plants.The short-term decrease in air temperature, occurring locally at the ground level, did not result in excessive cooling of the soil.Hydration of the surface soil layers at the start of the vegetation period effectively satisfied the water requirements of the plants.The weather conditions in April were also favorable for agriculture and promoted rapid growth of the plants.As a result of a cold spell in May, the pace of plant growth and maturation was slower.In June, the agrometeorological conditions were varied as they were in the remainder of the season.
Soil samples were collected from the rhizosphere at a depth of 40 cm.Nematodes were collected in two steps, according to the different types of plant-parasitic nematodes associated with three sources of samples: soil, roots, and stems.
Fresh roots and stems of Jerusalem artichoke were cut into segments having a diameter of 1 cm and weight of 20 g.The samples were placed in 100-µm sieves, immersed in water, and incubated for 5 days according to the method described by Baermann [17].After incubation, nematodes were collected separately from each sample (roots and stems).Nematodes from the soil samples were collected by centrifugation.A subsample of 100 cm 3 of soil from each sample was centrifuged.
Nematodes obtained were killed with heated 6% formaldehyde and processed in glycerin, according to the method described by Seinhorst [18].They were permanently fixed on slides.Nematode species were identified at the species level based on the morphological traits of male and female individuals following the identification keys developed by Brzeski [19] and Andrássy [20].Species dominance index C was calculated as follows: C = (Na N −1 ) × 100%, where: Na = number of samples containing a given species, N = total number of samples.
For every species found in each sample (consisting of specimens isolated from 100 cm 3 of soil by centrifugation, and individuals isolated on Baermann sieves), the following parameters were calculated: ■ population density (number of individuals in 100 cm 3 of soil or 20 g of the roots and stem), ■ frequency of occurrence (number of occurrences of a species out of the total number of 24 samples), expressed in %.

Results
The nematode species identified in the 24 soil, root, and stem samples from H. tuberosus L. plantations are presented in Tab. 1. Population density in 100 cm 3 of soil and the frequency of occurrence were determined for each species.Based on the values of index C, 22 species were classified as occasional, 10 species were classified as accessory (frequency of occurrence <20%), and three species were classified as dominant (frequency of occurrence >50%).
Tab. 1 Species of herbivorous nematodes collected in five plantations of Helianthus tuberosus L. in Poland.

Species Trophic type
Nematodes/100 cm A total of 35 herbivorous nematode species belonging to 17 genera were identified in the analyzed samples.The population densities of A. fragariae (Ritzema Bos, 1891) and Ditylenchus sp. were high, with up to 280 and 180 individuals, respectively, in 20-g samples of fresh stems.The pathogenicity of the above species will be analyzed in a greenhouse experiment in future.Aphelenchoides fragariae had previously been found to colonize the leaves of H. tuberosus L. in the Korean provinces of Gyeonggi and Gwangweo [15].The above species were extracted from infected tissues.The leaves of Jerusalem artichokes collected in Ujkowice, Wołczkowo, and Rumia exhibited characteristic symptoms of nematode infection, including brown discoloration of plant tissue, necrotic lesions, and large necrotic patches on leaves (Fig. 1).The observed symptoms were not associated with any fungi or bacteria.
Nematode vectors of viral diseases (a total of six species with frequency of occurrence of 4-28%) are deemed potentially harmful for plants.Half of the analyzed samples were colonized by Trichodorus viruliferus and Paratrichodorus pachydermus, which are able to transfer TOBRA viruses to the roots of several plant species.Three species of the genus Longidorus, capable of transmitting several NEPO viruses to other plants, were also identified: L. attenuatus (Fig. 2), L. elongatus, and L. leptocephalus (Tab.1).Ectoparasitic nematodes of the genera Mesocriconema, Paratylenchus, Bitylenchus, and Scutylenchus were characterized by small population densities that were unlikely to exert significant effects on plant yield.The population densities of endoparasitic nematodes of the genus Pratylenchus (Fig. 3) and semiendoparasitic nematodes of the genera    Helicotylenchus and Rotylenchus were low.Owing to their occurrence (28%) and high population densities, the ectoparasites Paratylenchus nanus and P. projectus should be monitored in future.Species of the families Aphelenchoididae and Anguinidae were identified on the basis of the fact that selected aphelenchids species are herbivores.Half of the analyzed samples contained two species of enthomoparasitic nematodes of the genera Mermis and Steinernema (a total of six specimens).However, bacteria-feeding nematodes play key roles in the decomposition of organic matter in ecosystems.In the analyzed samples, the following species of bacteria-feeding nematodes were identified: Cylindrolaimus de Man, 1880; Rhabditis sp.Dujardin, 1845; Plectus sp.Bastian, 1865; Anaplectus de Coninck and Schurmann Stekhoven, 1933; and Aporcelaimus sp.Their population densities reached up to 50 specimens in 100 cm 3 of soil, and their frequency occurrences were estimated at 50-75%.The observed changes in nematode communities in the rhizosphere of H. tuberosus L. plants indicate that further research is needed to explain the role of two species of Aphelenchoides and Ditylenchus sp., which colonized the stems of Jerusalem artichokes.They were classified as herbivores based on the presence of well-defined stylets.

Discussion
Helianthus tuberosus L. is generally susceptible to infections caused by fungi, bacteria, and tobacco mosaic viruses [26].It is also colonized by nematodes Caconema radicicola, Ditylenchus dipsaci, Aphelenchoides ritzemabosi, Heterodera marioni, Meloidogyne sp., and Puccinia helianthi [24].In this study, the analyzed samples of H. tuberosus L. were colonized by a total of 22 occasional species, seven accessory species, and three dominant species of nematodes (Tab.1).The members of the D. dipsaci complex cause serious infections in many plants grown in Europe.Ditylenchus gigas, for example, is pathogenic to broad beans [27].The specimens of Ditylenchus isolated from the stems of H. tuberosus L. require molecular analysis to confirm their species identities in a further study.Migratory endoparasites of the genus Pratylechus feed on cortical plant tissues, causing necrotic changes in roots and leading to plant death.The results of this study and published data indicate that A. fragariae is a ubiquitous pathogen in H. tuberosus L. Aphelenchoides fragariae infects stems and leaves by migrating to the stem and entering the stomata.The analyzed stems and leaves were significantly (20%) contaminated with A. fragariae (Fig. 1).The initial symptoms of infection in Jerusalem artichoke leaves were irregular, water-containing lesions between leaf veins, which spread gradually and caused necrotic changes in plant tissues -symptoms that are very similar in both infested Jerusalem artichoke [15] and in other hosts, for example silver birch [28], among diverse ornamental plants [29][30][31].The genus Aphelenchoides Fischer, 1894 is composed of ubiquitous fungi-feeding species, as well as species that infect insects and plants.These nematodes feed on both fungi and plants [32].Numerous plant species are colonized by the foliar nematodes [28] that are the most widespread parasitic nematodes in Poland.Nematodes of the genus Aphelenchoides are usually transmitted via clothing, footwear, containers, packaging (wood), land vehicles, paper, soil, sand, and gravel [33].According to the Center for Agriculture and Biosciences International (CABI), Hirsutella rhossiliens is a natural enemy to Aphelenchoides; however, it can also be used to fight Ditylenchus dipsaci, Globodera pallida, Heterodera avenae, and H. schachtii [26].
This study has revealed that six nematode species could be vectors of viral diseases in the Jerusalem artichoke in Poland.The greatest threat is posed by Trichodorus viruliferus (vector of several TOBRA viruses), characterized by a high frequency of occurrence (28%) and high population density, as well as by Longidorus attenuatus, which commonly occurs in Polish soils (Fig. 2) [14,25,34,35].The ectoparasite feeds on roots, and it can transmit NEPO viruses to healthy plants.The prevalence of nematode vectors of viral plant diseases suggests that viral symptoms in leaves should be closely monitored, and that virology tests should be performed to detect latent viruses in H. tuberosus L. plantations [36]
. © The Author(s) 2018 Published by Polish Botanical Society Acta Soc Bot Pol 87(2):3578 Zapałowska and Skwiercz / Populations of parasitic nematodes colonizing Jerusalem artichoke Conclusions ■ Further research is required to monitor the spread of parasitic nematodes colonizing the rhizosphere of H. tuberosus L. in long-term monocultures.■ The roles of two unidentified species of Aphelenchoides sp. and Ditylenchus sp. in colonizing H. tuberosus L. stems should be determined using seedlings grown in a pot experiment under in vitro conditions.■ Six nematode vectors of viral diseases were identified in 40% of the 24 analyzed samples, and further research is needed to identify the causes of viral diseases in plants, in particular in H. tuberosus L.