Aphelenchoides varicaudatus (Nematoda: Aphelenchoididae) and Helicotylenchus erythrinae (Nematoda: Hoplolaimidae) from Garlic Plantation in Magelang, Central Java, Indonesia

Many nematodes can be associated to garlic plants. Twenty genera of nematodes have been recorded to be associated with the roots of garlic plants in Yemen; Antarctenchus, Aphelenchoides, Aphelenchus, Basiria, Boleodorus, Ditylenchus, Helicotylenchus, Heterodera, Hoplolaimus, Meloidogyne, Microposthonia, Nothanguina, Pratylenchus, Rotylenchulus, Rotylenchus, Scutellonema, Tetylenchus, Tylenchorhynchus, Tylenchus, and Zygotylenchus found in garlic (Mohamed, 2015). While in Indonesia, from 2019 to 2021, several plant parasitic nematodes have been found accompanying garlic plants in Central Java and East Java, Indonesia, such as Helicotylenchus, Aphelenchoides, Rotylenchulus, Aphelenchus, Criconemoides, and Tylenchus (Wulandari & Indarti, 2019; Wulandari et al., 2021; and Kusuma et al., 2020). Several species of plant parasitic nematodes have wide host range, such as Ditylenchus dipsaci (Musyarofah & Indarti, 2019). Amritha and Budijastuti (2018) stated that Ditylenchus dipsaci, Aphelenchoides sp. and Xiphinema sp. were found in imported and local garlic in East Java. Therefore, identification of the most common plant parasitic nematode associated with garlic is needed for further management and avoid yield losses on garlic.

The most common nematode genera associated with garlic plantations in Magelang, Central Java, were Aphelenchoides and Helicotylenchus (Wulandari et al., 2021). The taxonomic determination of Aphelenchoides at the suborder level is quite controversial between “Tylenchid” and “Aphelenchid” (Sanchez-Monge et al., 2015). Many species are not fully described and do not have molecular data for taxonomic studies (Zhao, 2006; Sanchez-Monge et al., 2015). Some species of Helicotylenchus have very similar diagnostic characters, and boundaries between species are not well established (Subbotin et al., 2015). The existence of complex species in the genera makes them morphologically almost indistinguishable but may be quite distant from each other by their phylogeny (Subbotin et al., 2015). The combination of morphological and molecular identification shown to be most effective method for species identification (De Ley et al., 2005)

Nucleotides data for Aphelenchoides and Helicotylenchus genera are mostly limited in The National Center for Biotechnology Information (NCBI). At the time this research was conducted, there were only 1237 and 758 nucleotides records for Aphelenchoides and Helicotylenchus, consecutively (NCBI accessed in November 2021). However, molecular data at species level are obviously rare. Several molecular studies on Aphelenchoides were reported in Indonesia, such as A. besseyi on rice (Rahman et al., 2018; Sembiring et al., 2019) and A. varicaudatus on garlic plants (Kusuma et al., 2020), but there was no information about the molecular study of Genus Helicotylenchus in Indonesia. This study aimed to characterizing the most dominant nematodes from garlic plants in Magelang, Central Java, Indonesia, based on morphological characters and DNA sequences.

For this study formal consent is not required.

Common symptoms caused by nematodes include yellowing, stunted growth, and accompanied by a decrease in yield. Plant damage due to nematodes alone is difficult to observe because the aboveground symptoms are similar to symptoms of nutrient deficiency and they also can be associated with other pathogens. It is necessary to carry out laboratory tests to see the presence of important plant-parasitic nematodes that can potentially reduce yields (Schmitt & Sipes, 2000; William et al., 2018). Aphelenchoides found in each extracted part (roots, bulbs, soil) in each field surveyed. The highest population for Aphelenchoides was found in the first field, which showed severe bulb rot (causing soil cover up the bulbs) with the presence of white fungi. The presence and interaction between nematodes and fungi in plants could be antagonistic interactions or synergistic interactions. Antagonistic interactions can occur through competition for space and food. The synergistic interactions can result in more severe damage to the host plant (Zhang et al., 2020). In other hand, Helicotylenchus mostly found from the soil for both field.

The Aphelenchoides samples had characters as described by Huang et al. (2012) as A. varicaudatus. This species with a rounded set-off head, multireflexed or outstretched ovary, posterior ovary with V%=±60, conoid convex tail with variable tail terminus shape, one or two mucronate structures on the tail terminus (Huang et al., 2012). Aphelenchoides sample from Magelang have the same body length range as A. varicaudatus Yunnan, China.

The molecular analysis supported Aphelenchoides Magelang Indonesia as A. varicaudatus species. All sequences comparison in this study were picked from large subunit (LSU) 28S rRNA gene region. This region has conserved domains and expansion segments, such as the D2–D3 expansion region. This region is useful for designing of diagnostics tool for the identification of nematodes to the species level (Cunha et al., 2018). In this study, A. varicaudatus Magelang Indonesia separated from unidentified Aphelenchoides species (Aphelenchoides sp.) from various country based on its 28S gene region. A. varicaudatus from Yunnan, China is closely related to Aphelenchoides sp. from ginger plant (Zingiber sp.) in Indonesia from its small subunit (SSU) and very different to A. besseyi and A. fragariae (Huang et al., 2012). The phylogenetic tree in this study also revealed that A. varicaudatus separated from other species of Aphelenchoides such as A. besseyi, A. fragariae, A. ritzemabosi based on 28S region as Huang et al. (2012) reported.

Morphology and molecular identification of Aphelenchoides from garlic plants in Magelang showed the species indicated to A. varicaudatus. A. varicaudatus is not a common plant-parasitic nematode reported species from Genus Aphelenchoides like A. besseyi, A. fragariae, and A. ritzemabozi. The status of A. varicaudatus was still unclear. However, study about this species is also important, remembering that this species was also found in the rose root and pine xylem (Huang et al., 2012; Ibrahim & Hooper, 1994). A study by Santos et al. (2021) revealed that Aphelenchoides pseudogoodeyi, which also has unclear information about its pathogenicity, is an opportunistic pathogen as the injury was required to induce symptoms in Bird's-Nest Fern and Oriental Lily. It was showed that a mainly mycophagus Aphelenchoides could become phytophagous under stress conditions (Santos et al., 2021). A. varicaudatus was first described by Ibrahim and Hooper (1994) from rose roots plants. Furthermore, A. varicaudatus was found in Yunnan, China from Simao Pine plant (P. kesiya var. langbianensis) (Huang et al., 2012). A. varicaudatus could be found in bark/moss/lichens and able to grow on fungi media (Sanchez-Monge et al., 2015). This indicated that A. varicaudatus has a wide distribution and different host associations (Huang et al., 2012). The species in Aphelenchoides are very diverse and have different types of feeding. Most species in Aphelenchoides are mycophagous (Kanzaki, 2012). There are 13 other species known as plant parasites. Most of them are generalists because they have a broad host range (Sanchez-Monge et al., 2015). Only a few species are economically important (Nickel & Hopper, 1991).

The morphological characters of Helicotylenchus are quite complicated to observe between species. Our sample had descriptions as H. erythrinae in Uzma et al. (2015) based on lips and tail observations and also description by Riascos-Ortiz et al. (2020). Based on morphometrical data, our specimens close to H. erythrinae from Calarcá, Quindío, Colombia by Riascos-Ortiz et al. (2020) with high variability observed in some characters. The existence of intraspecific variation and limited character information makes identification difficult. However, no male nematodes were found in this study. According to Riascos-Ortiz et al. (2020), female H. erythrinae species have functional spermatheca with sperm in it and there are male nematodes. Species H. erythrinae has a loose spiral post-mortem habitus, a hemispherical lip region with an indented or flattened anterior knob, a tail with long ventral projections (Riascos-Ortiz et al., 2020) which these morphological characteristics were found in our specimens.

The phylogenetic tree supported that Helicotylenchus from Magelang (OL757653) clustered well with H. erythrinae (MT321739) from Colombia. Percent identification with this species was only 95.66 %, indicating the Magelang specimen had some nucleotide variations with H. erythrinae (MT321739). However, there was only one sequence submitted for H. erythrinae out of a total of 758 submitted sequences in the Genus Helicotylenchus (data accessed in November, 2021). Nucleotide sequence observations showed specific characters with H. erythrinae (MT321739) at some orders of nucleotides length, such as at 56 – 66, and 78 – 89 of 569 nucleotides compared with other representative species in the Genus Helicotylenchus.

Based on morphological observations and molecular characters, Helicotylenchus found from garlic plantations in Magelang, Central Java, refers to the H. erythrinae species. Molecularly, H. erythrinae was first reported by Riascos-Ortiz et al. (2020) from banana plantain in Colombia. H. erythrinae has a fairly wide host range, including dadap (Sher, 1966), pepper, ginger (Koshi et al., 2005), tea (Nalini et al., 2005), cocoa, olive (El-Borai & Duncan, 2005), cassava, rice (Bridge et al., 2005). In addition to being found in some cultivated plants, H. erythrinae was also found in uncultivated jungle soils (Sauer & Winoto, 1975). H. erythrinae, H. dihystera and three undescribed Helicotylenchus species were found from Java (Sher, 1966). Systematics within the genus of Helicotylenchus is in a very unsatisfactory state and identification to species is difficult (Wouts & Yeates, 1994).

Future study for A. varicaudatus and H. erythrinae found from garlic plantation is needed since the sequences data in NCBI for both species were limited. Molecular characterization from other DNA genes target is needed to perform more comprehensive molecular characterization.