Phytonematodes Community and Polyphasic Character of Aphelenchoides varicaudatus on Garlic Plants in Tegal Regency, Central Java

The interception of Ditylenchus dipsaci, D. destructor, and Aphelenchoides fragariae, which are classified as quarantine pests, has been reported to occur on imported garlic bulbs used for consumption at several traditional markets in Bogor. This finding should increase awareness of the chance of garlic contamination in Indonesia’s garlic fields. This study aimed to evaluate the nematode community and determine polyphasic characters of Aphelenchoides species from garlic plantations. The study was conducted by sampling soil and plants from garlic plantations in Tuwel and Rembul Villages, Bojong District, Tegal Regency, Central Java in December 2018. Nematode extraction was done using a mist chamber for plant materials and sugar flotation – centrifugation methods for soil samples. Parameters measured included nematode species identity, absolute population and prominence indexes. Nematode identification was conducted based on their morphological characters. Further identification based on morphometric and molecular characters (polyphasic) conducted for nematodes species suspected as quarantine pests. Six generas of nematodes identified were Helicotylenchus, Aphelenchoides, Rotylenchulus, Aphelenchus, Criconemoides, and Tylenchus. Aphelenchoides sp. had the highest prominence value from plant tissue with a value of 6.32 and is categorized as a quarantine pest genus (A. fragariae) in garlic and was further identified to the species level. Further identification based on polyphasic characters showed the Aphelenchoides sp. found was A. varicaudatus.


INTRODUCTION
Indonesia imports more garlic compared to its national production. Within 2007Within -2018 percentage of national needs were imported, which is approximately reach 446,522.20 kg (BPS, 2019). This high importation cause high risk of quarantine pest to enter Indonesia. Nematode inception reports have shown occurences of Ditylenchus dipsaci, D. destructor, and Aphelenchoides fragariae on imported garlic bulbs used for consumptions and sold in traditional markets located in Bogor and surrounding areas. These nematodes are quarantine pests (Kementan, 2018) on garlic and threat garlic production of high altitude fields in Indonesia. Damage due to nematodes are estimated to reduce 10-100% of garlic production in various countries (Sturhan & Brzeski, 1991;Abawi et al., 2012;Cheng et al., 2015).
Yield loss in garlic may occur when the fields are contaminated with nematodes. Nematode contaminated seeds are possible threats for crops. In addition, nematodes are able to reproduce on plants, which posses a larger threat for garlic production. Garlic seed importation by Indonesia has been reported during 2007-2009 with an average volume of 298,323 kg and 2017-2018 with an average volume of 1,000,350 kg (BPS, 2019). Garlic seed importation should be monitored for the possibility of quarantine nematodes to enter Indonesia. Nematode species identification with a comprehensive approach using morphological, morphometric, and molecular techniques, or known as polyphasic characteristics, are considered to provide accurate results. Biochemical and molecular methods and their tools have successfully identified nematodes (Carneiro et al., 2017). Therefore, these methods are hoped to confirm nematode species characteristics accurately.
Nematode contamination in fields sets a threat of yield loss and spreading to other garlic production fields. Correct identification to species levels will provide correct information regarding to nematode species. Therefore, identification of nematode community in garlic productions should be done in fields using polyphasic methods.

Location and Sampling Techniques
Survey and sampling were done using a purposive sampling method (van Bezooijen, 2006). Garlic plants that were suspected to be infected by nematodes (Indarti et al., 2018) were obtained from a garlic production field in Village of Rembul and Tuwel, Bojong, Tegal.

Nematode Extraction
Nematodes were extracted from 100 mL of soil sample solutions using a floatation and centrifuse method (van Bezooijen, 2006). Nematode extraction from plant parts (10 g of root, bulb, stem, and leaves) was done using a Baermann funnel and modified with a mist chamber (EPPO, 2013). Nematode specimens were prepared using methods from Ryss (2017).

Morphological and Morphometric Identification
Nematode specimens were identified to genus and species using identification keys form Tarjan et al. (1977). Identification was done under a binocular microscope (Olympus BX 51). Density of nematode populations frim each samples were counted at enlargement of 40×. Morphometric characteristics of nematodes were determined using de Man's formula.

Data Analysis
Data analysis were done for soil and plant tisuue samples. Absolute population (AP) were caluculated using the following formula: Absolute frequence (AF) was calculated from the number of samples that contained a certain nematode species divided by the total number of samples tested. Population density and absolute frequency were used to calculate prominence value (PV) (Norton, 1978;Fourie et al., 2001), using the following formula: Kusuma et  AP : nematode population from each 100 g soil sample suspension or 10 g of plant parts. n1 : average number of nematodes counted from v1. v1 : is the suspension volume (mL) measured in counting dish. v2 : total volume (mL) of sample suspension extracted. v3 : volume (mL) of soil suspense or weigth of plant tissue (g). (2)

DNA Extraction and Amplification
Nematode DNA was extracted using worm lysis buffer method from an individual nematode (Holterman et al., 2006). Results from DNA extraction was analyzed using a PCR and a universal D2A primer (5´-ACAAGTACCGTGAGG-GAAAGTTG-3´) and D3B primer (5´-TCGGAAGGAAC-CAGCTA CTA-3´) to amplify the D2-D3 gene segment of 28s rRNA (Subbotin et al., 2006). PCR products were sequenced and compared with GenBank, NCBI to determine homology and query cover value.

Sequencing and Nucleotide Analysis
PCR amplification results were sent to 1 st Base (Malaysia) to do a nucleotide sequencing. Sequence were analyzed using program basic local alignment search tool (BLAST) on national center for biotechnology information (NCBI). Phylogenetic trees were produced using Mega version 7.0.26 (Kumar et al., 2016) based on the maximum likelihood. Bootstrap were analyzed with 1,000 replications to reconstrute phylogenetic tree.

Symptoms Description
Garlic plants from Tegal showed several symptoms suspected to be caused by nematodes. Symptoms that occurred above ground were yellowing, stunted, falling leaves, and leaf distortion. Symptoms found on organs below ground were fewer numbers and damage on roots ( Figure 1). Symptoms found from Tegal samples were similar to damage symptoms on garlic caused by nematodes (Shurtleff & Averre, 2000;Schwartz & Mohan, 2008;Indarti et al., 2018). Garlic plants infected by parasitic nematodes counld not stand upright, stunted, had yellow leaves, thickened and short leaves, falling leaves, had yellow or brown spots, softening stems, and death of plants.

Garlic Nematode Community
Nematodes recovered from roots and garlic bulbs are shown in Table 1. Nematodes found in this study were associated with garlic and other vegetables (Firake et al., 2016;Keshari et al., 2018). This indicates that plants and rizhophere of garlic plants in Tegal are not infested by the quarantine pest Ditylenchus sp.
Based on population densities, Aphelenchoides sp. showed the largest population of 10 nematodes in 10 g of bulb and healthy plants (  (Table 2). Several reports have demonstrated that nematode population of this secies dominates nematode communities on vegetable crops due to their parthogenetic reproducing ability (Firake et al., 2016;Mathivathani & Subramanian, 2018).

Aphelenchoides sp. Morphologic and Morphometric Characteristics
Female Aphelenchoides sp. from this study had round and offset heads, and basal knob shaped stylets. Metacorpus were fully elongate shaped. Valves were located at the body's posterior. Tails were dorsally convex conoid, tapered, and showed a unique mukro on the tip of the tail (Figure 2). Morphological and morphometric characteristic of all Aphelenchoides sp. isolates demonstrated similarity with A. varicaudatus as described by Huang et al. (2012), (Table 3).
The tail characteristics and number of lateral lines of Aphelenchoides sp. within this study were different between A. fragariae and A. besseyi (Huang et al. 2012;EPPO, 2017). The number of lateral lines on Aphelenchoides sp. specimen were similar to A. besseyi that was 4, while A. fragariae only have 2 lines. A. fragariae and A. besseyi tails were elongate-conoid and conoid, while Aphelenchoides sp. tails were convex-conoid. Therefore, characteristics showed by Aphelenchoides sp. specimens were similar to morphological characteristics of A. varicaudatus as described by Huang et al. (2012).

Aphelenchoides sp. Molecular Characteristics Based on D2-D3 Sequence on 28S rDNA Gene
PCR amplification on 28S rDNA gense region D2-D3 using D2A and D3B primer, DNA isolate resulted in a single band ± 780 bp ( Figure 3). Based on the highest identity and query cover score and the lowest E value, Aphelencoides sp. (MN587128) from this study showed similarity to A. varicaudatus (Table 4). Phylogenetic analysis showed that Aphelenchoides sp. had similarity to A. varicaudatus from Pinus kesiya (Huang et al., 2012) with 100% homology (Figure 4).

CONCLUSION
Parasitic nematodes collected from garlic samples were Aphelenchoides sp., Helicotylenchus sp., Rotylenchulus sp., Aphelenchus sp., Tylenchus sp., Criconemoides sp., while non-parasitic nematodes recovered was Dorylaimus sp. Aphelenchoides sp. had the highest prominence values from plant tissue ca. 6.32 and one species of this genus is a quarantine pest. Further identification based on the polyphasic characteristics show that the Aphelenchoides sp. specimen was A. varicaudatus.

ACKNOWLEDGEMENT
The author would like to thank the Agency for Extension and Agricultural Resource Development (BPPSDMP) which has funded this research.