BIODIVERSITAS ISSN: 1412-033X

Volume 21, Number 5, May 2020 E-ISSN: 2085-4722

Pages: 2068-2073 DOI: 10.13057/biodiv/d210533

Short Communication:
First record of Hirschmanniella mucronata (Nematoda: Pratylenchidae)
in Yogyakarta, Indonesia

SIWI INDARTI1,2,♥, ALAN SOFFAN1,♥♥, MUHAMMAD MAULANA FARDANI ANDRASMARA1

1
Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada. Jl. Flora, Bulaksumur, Sleman 55281, Yogyakarta, Indonesia.
Tel.: +62- 274)-563062, 901290, Fax.: 62-274-563062, 519717, ♥email: siwi.indarti@ugm.ac.id; ♥♥alan.soffan@mail.ugm.ac.id
2
Agrotechnology Innovation Centre, Universitas Gadjah Mada. Jl. Tanjung Tirto, Kalitirto, Berbah, Sleman 55573, Yogyakarta, Indonesia

Manuscript received: 2 October 2019. Revision accepted: 18 April 2020.

Abstract. Indarti S, Soffan A, Andrasmara MMF. 2020. Short Communication: First record of Hirschmanniella mucronata (Nematoda:
Pratylenchidae) in Yogyakarta, Indonesia. Biodiversitas 21: 2068-2073. Hirschmanniella spp. is one of the worldwide plant-parasitic
nematodes affecting major losses in rice production and impact up to 25% yield losses especially on irrigated rice. Infection of
Hirschmanniella spp. on the root system leading to the typical symptom of red color in the rice rooting system. In order to identify the
species variation of Hirschmanniella spp. from the collected samples in Yogyakarta Province of Indonesia, a molecular-based
identification using Polymerase Chain Reaction (PCR) method was conducted, complemented by morphological identification
technique. PCR based identification was carried out by amplifying the area of 28S rRNA using universal nematode primer (D2A / D3B)
which resulting about 766 bp of amplicon. Blastx analysis from Genbank showed that Cangkringan sample confirmed to be H.
mucronata species, while other samples from Banguntapan and Imogiri were H. oryzae species. The Cangkringan samples of H.
mucronata have 97.5 % similarities with Belgium sample, forming separate clades with other samples. While both Banguntapan and
Imogiri samples have the 99 % similarity with H. oryzae and were located in the same clade, but separated from Cangkringan sample.
Morphological identification confirmed both species were distinctly based on the unique characters of the tail tips. H. mucronata
therefor is the first report nematode species in Indonesian rice field. Precaution should be designed to prevent the potential distribution
of H. mucronata to other areas.

Keywords: Hirschmanniella mucronata, H. oryzae, molecular identification, Yogyakarta

INTRODUCTION Meloidogyne sp., and H. oryzae (Bridge et al. 2005). With

the increasing movement of the rice grain and peoples
Rice (Oryza sativa sativa) is one of the most important across locations, Indonesian rice fields will be vulnerable
crops, and more than half of the world’s population have for the infestation of other species in addition to
been using rice as a staple food (Zin Oo and Hla Maw Meloidogyne spp. and H. oryzae. Genus Hirschmanniella is
2016). Indonesia is one of the biggest rice consumers due known to be one of the most important nematode genera on
to its role as the main staple food. In order to meet the rice (Khun et al. 2015), which can caused rice yield loss
increasing rice demand, Indonesian government has from 12.5% up to19 % (Bala and Khan, 2004). Two or
implemented various technology such as through SRI more species of Hirschmaniella usually found in the same
(System of Rice Intensification). While intensification area and may lead to severe damage (Khan and Bala 2003).
effort significantly increases rice production, however, at On the regular survey of nematode infestation across
the same time, it deploys the potential risk, which becomes different areas in Indonesia, particularly in Yogyakarta,
a significant threat to rice production. One of the potential Java island, we finally reported the first occurrence of
risks is due to nematode infestation. The plant-parasitic nematodes species Hirchmaniella mucronata which
nematodes play an important role and account for yield previously had not been reported yet. This report serves as
losses to the extent of 90% (Jain et al. 2012). There are an important alert for the potential infestation in the near
several plant-parasitic nematode species known being able future which might affect the decreasing of Indonesian rice
to infest rice plant, including white tip (Aphelenchoides production.
besseyi), rice root-knot (Meloidogyne graminicola), rice
root (Hirschmanniella spp.), rice stem (Ditylenchus
angustus), rice cyst (Heterodera oryzicola), root lesion MATERIALS AND METHODS
(Pratylenchus spp.), Hoplolaimus, Criconemoides, and
Tylenchorhynchus (Swe 1997; Than 2003). Nematode sampling and extraction
The distribution studies of those species across the Samples were collected from the root system with
Indonesia rice fields are limited. Many studies reported that nematode symptoms in 2018. Sampling locations were
Indonesia rice fields mostly have severe damage due to mainly conducted in the rice production areas of
 INDARTI et al. – First record of Hirschmaniella mucronata in Yogyakarta 2069

Yogyakarta, Indonesia, namely Cangkringan, Banguntapan, and aquabidest 2.5 µL, 2.5 µL and 5 µL, respectively. PCR
dan Imogiri. Soil samples were extracted for nematode program was performed with annealing, denaturation and
presence using the modified Whitehead Tray method extension temperature were 95°C, 54°C, and 72°C,
(Southey, 1986). Before the nematode analysis, the root respectively, with the time period of 30, 30, and 15 sec.,
samples were scored to categorize the root necrotic respectively. The final extension was performed at 72oC for
damage. Modified scoring of root damaged was grouped 10min, followed by 4oC. The PCR products were run on
into 10 classes according to the percentage of root cortex 2% agarose gels and visualized on UV transilluminator.
surface covered by necrotic surface, 0: no necrotic on the The amplified PCR products within the expected size range
root cortex, 1: 1-10 % root cortex covered by necrotic, 2: were gel purified and sequenced on ABI 3500 genetic
11-20 % root cortex covered by necrotic, 3: 21-30% root analyzer (Life Technologies). Sequences were analyzed
cortex covered by necrotic, 4: 31-40 % root cortex covered using Bioedit ver. 7.1.9 followed by BLASTx searches
by necrotic, 5: 41-50 % root cortex covered by necrotic, 6: conducted in the NCBI database, to confirm the species
51-60% root cortex covered by necrotic, 7: 61-70%’ 8: 71- name. Finally, the phylogenetic tree of the 28S rRNA was
80%, 9: 81-90%, and 10: 91-100% (Coyne et al., 2007). constructed using the maximum likelihood statistics with
Nematode extractions from root samples were done by the LG+G best fit model to look at the relationship with the
cutting the root samples into 1 mm pieces then were laid on available database.
the filter paper on the nylon screen supported by the
modified bucket allowing the water to attach the root Morphological identification
samples. The dipping was at room temperature for 24 h. The morphological characters of Hirschmanniella spp.
This method referred to Whitehead Tray Technique were evaluated using light microscope Olympus CX 31
(Southey 1986) with modification. The nematode which with magnification 40-1000×. The identification of
was swimming out to the water was collected on the tubes specimens was based on the related literature, i.e., Ebsary
for further both morphological and molecular-based and Anderson (1982) and Loof (1991). The main characters
identification. Morphometric measurement was for morphological identification are a general shape of
incorporated to support identification in species level. nematode and their body length, cephalic framework, type
and position of esophagus to intestine (Berliner et al.,
DNA extraction 2017). According to available literature, the identification
Modified CTAB methods were used to extract the DNA up to species level can be distinguished from one species to
from the nematode samples (Zhou et al., 2007). In brief, the another by the shape of the tail as in Figure 1.
four adults Hirschmanniella spp. were picked up and each
nematode than chopped into 4-5 pieces by using tips of Morphometric measurement
needle followed by the addition of 25µL buffer CTAB in a Morphometric characters were analyzed using De Mann
1.5 mL tube. The samples were incubated in 65oC formula (Southey 1986). Drawing and measurement of
temperature for 30 min with every 10 mins samples were each specimen were conducted using a camera lucida
up and down prior to centrifuge for 5min with 5000 rpm. which was connected to microscope Olympus BH2. Main
Chloroform: Isoamylalkohol (CIAA) with ratio 24:1 were notation of De Mann formulae includes n (number of
added to the sample followed by gentle shaking and 15 min specimens), L (total body length in nm or µm), a (body
centrifuge at 10.000 rpm, room temperature. Finally, the length: greatest body width), b (body length: distance from
supernatant was collected and mixed with cold absolute anterior end to junction of esophagus and intestine), V
alcohol with a ratio of 1:2 followed by 24 hours incubation (distance of vulva from anterior end X 100: body length),
at-20oC. Sample washing was done by using 70% cold and length of female tail (distance from anus to tip tail).
alcohol after 10.000 rpm for 15min after previous
incubation. The final template of DNA was obtained by
10.000 rpm for 15min centrifuge followed by TE dilution RESULTS AND DISCUSSION
for 20 µL. The DNA concentration was measured using
Nanodrop spectrophotometer (Thermos, USA), and the Hirschmanniella spp. infect and multiply in the root
samples were maintained at -20oC until PCR and system, through the air space across the radial lamella. An
sequencing. infected root system becomes necrotic, followed by a
change in color to red and brown, indicating the decay
Amplification and sequencing process. This typical symptom is due to the injury of the
The D2A-D3B primers (D2A: 5’ACAAGTACCGTGA cortex of the root system (Bridge and Starr 2007). The rice
GGGAAAGTTG 3’; D3B:5’TCGGAAGGAACCAGCT root systems (Figure 2) infected by Hirschmaniella spp.
ACTA 3’) with 766 bp expected product were used to were stunted in growth, which could lead to rice production
amplify 28S rRNA of nematodes which previously had failure (Figure 3). Details of the Hirschmaniella spp.
been reported to distinguish until species level (Nunn infestation is presented in Figure 4.
1992). PCR Kit (Redmix BioLine) was utilized with a total
volume of 12.5 µL, including the DNA template, primers,
2070 B I O D I V E R S I T A S 21 (5): 2068-2073, May 2020

Figure 1. Tail shape of Hirschmanniella spp. A. H. gracilis, B. H. spinicaudata, C. H. oryzae, D. H. caudacrena, E. H. miticausa, F. H.
shamim G. H. mucronata (Loof 1991)

An additional observation was conducted to correlate

the injury level, Hirschmanniella spp. population, rice
cultivar, and the location site, as presented in Table1. It
was shown that the highest injury level occurred in the
Cangkringan, Sleman on both Ciherang and Mentik Wangi
cultivars, and it had a positive correlation with the number
of Hirschmanniella spp. in the rooting system. While the
IR 64 cultivar had less injury in the same location. Both of
these cultivars, Ciherang and Mentik Wangi in the same
area, were also as susceptible as IR64 in different locations.
Based on this result, it was obvious that rice cultivar and
site location seem to have a significant contribution to the A B
injury level and Hirschmanniella spp. population. It was
suggested that soil abiotic factors, especially soil humidity,
Figure 2. Infected root of the rice by Hirschmanniella spp (A)
C organic contents, and temperature have a significant
and healthy root (B)
effect on nematode abundance. Report of Mutala’liah et al.
(2018) showed that cultivar host plants influenced the
plant-parasitic nematodes abundance and Mutala'liah et al.
(2017) also reported that C-Organic positively affects
nematodes abundance, including on root-lesion nematode
that belongs to the same family with Hirschmanniela.

Tabel 1. Hirschmnniella sp. population and the root damage

index based on location and rice cultivar

Root Hirschmanniella
Local rice
Location damaged spp. population
cultivar
index (per gram root)
Cangkringan, IR64 2 14
Sleman Ciherang, 6 134
Mentik wangi 6 126
Banguntapan, Ciherang 4 45
Bantul
Imogiri, IR64 5 69
Bantul Figure 3. Field symptom of the Hirschmnniella spp. infestation.
The rice plants in inside the circle with stunted growth and
flowering were delayed
 INDARTI et al. – First record of Hirschmaniella mucronata in Yogyakarta 2071

A B C

Figure 4. Root infected symptom by Hirschmanniella spp. in Imogiri (A), Banguntapan, (B), and Cangkringan (C)

M A B C bootstraps. The phylogenic tree revealed that the Imogiri

and Banguntapan samples were in the same clade together
with H. oryzae from Myanmar, Iran, Vietnam, and the
Philippines. The Cangkringan samples were grouped in
another clade recognized as H. mucronata, together with H.
mucronata from Belgium, the Philippines, and South
China.
In general, Genus Hirschmanniella is a member of the
family Pratylenchidae, which has monomorphism
character, both either female and male are vermiform and
slender and easily identified by their long body (1 to 4
mm). They have colorless body with well-developed
cephalic framework (head) and consisted of one short
feeding apparatus called stylet with rounded knobs. They
have esophageal gland, which overlapping over the
intestine in the ventral side (Berliner et al., 2017). All
criteria were found on all specimens of Hirschmaniella
from Cangkringan, Imogiri and also Banguntapan.
However, there were differences in female tip shape of
Figure 5. Amplicons of ±766 bp were resulted from A.
Cangkringan. B. Banguntapan. C. Imogiri )
Cangkringan specimen to both Imogiri and Banguntapan
(Figure 7).
The morphological observation of the female tail shape,
Molecular detection of samples collected from Imogiri, especially on the tail tip confirmed that the Cangkringan
Banguntapan, and Cangkringan was conducted by PCR sample was H. mucronata, whereas the Banguntapan and
using the D2A/D3B primer. All of the samples resulting in Imogiri samples were H. oryzae. H. mucronata is
766 bp amplicons (Figure 5). The sequencing results were characterized by having the irregular tail edge sharpened
submitted to GenBank (https://www.ncbi.nlm.nih.gov/) into a projection (Khun et al. 2015), while H. oryzae has a
under Accession numbers of MK205364, MK2419389, and regular tail shape with an outside projection (Luc and
MK241938 for H. oryzae Imogiri, H. oryzae Banguntapan, Goodey 1964) (Figs. 7-8).
and H. mucronata Cangkringan respectively, Identification based on molecular, and morphological
To evaluate the similarity of the three tested samples data confirmed the presence of two species of
with other reported Hirschmanniella spp. from the Hirschmanniella, which were H. mucronata (found on
GenBank database, multiple alignment sequencing was Cangkringan) and H. oryzae (found on Imogiri and Bantul).
conducted using the ClustalW program. The analysis And based on the morphometric data, especially some
showed that the Cangkringan sample had 84%-97% characters on the length of stylet, and a and b values were
similarity with H. mucronata from Belgium. The rest of the supported that specimen from Cangkringan is H.
samples from Imogiri and Banguntapan had 99% similarity mucronata (Table 2). Therefore, H. mucronata is the first
with H. oryzae. A phylogenetic tree (Figure 6) was report of this species infecting rice fields in Indonesia. This
constructed using MEGA6 with the neighbor-joining report should be followed up by anticipating the distribution
method, the Kimura 2 parameter model, and 1,000 of H. mucronata to other locations in Indonesia.
2072 B I O D I V E R S I T A S 21 (5): 2068-2073, May 2020

Figure 6. Neighbor-joining phylogenetic tree of 28 srRNA from nematode samples collected from rice fields in Cangkringan,
Banguntapan, and Imogiri. The best-fit Kimura 2 parameter model with 1,000 bootstraps was used to construct the tree with other
available Hirschmanniella spp. from the Genbank NCBI database

A B C

Figure 7. Female tail end of Hirschmaniella mucronata (from Cangkringan). (A) and H. oryzae from Imogiri (B) and Banguntapan (C)

A B C D

Figure 8. Anterior part showing lip, knob stylet, and female tail of Hirschmanniella. A-B. H. mucronata (from Cangkringan), C-D. H.
oryzae (from Banguntapan)
 INDARTI et al. – First record of Hirschmaniella mucronata in Yogyakarta 2073

Table 2. Morphometric data of the Hirschmanniella spp. females from Yogyakarta Indonesia (n = 5)

Reference (Khun et al. 2015)

Characters H. oryzae H. mucronata
H. oryzae H. mucronata
Length of body (µm )* 550-1,150 760-1,050 1,200-2,700 900-1,850
Length of stylet * 15-20 22-23 13-21 µm 20-29 µm
a 29.33-42 33-52.27 29-80.6 44.8-84
b 6.9-13.8 9.2-15.8 6.8-14 9.3-16.6
V 40-65 55-60 47.2-63.1 47.2-63.1
Length of tail* 80-85 60-90 45-123 µm 60-160 µm
Note: *) Measurements in µm

ACKNOWLEDGEMENTS Khan M, Bala S. 2003. Morphometric variations in Hirschmanniella

mucronata and H. oryzae from West Bengal. Intl J Nematol 13: 225-
228.
The authors thank the Faculty of Agriculture, Khun K, Decraemer W, Couvreur M, Karssen G, Steel H, Bert W. 2015.
Universitas Gadjah Mada, Yogyakarta, Indonesia for the Deceptive morphological variation in Hirschmanniella mucronata
research funding and Ayu Suci Wulandari (assistance on (Nematoda: Pratylenchidae) and a polytomous key to the genus.
Nematology 17: 377-400.
Nematology, Plant Protection Department, Faculty of Loof, P. 1991. The family Pratylenchidae Thorne, 1949. In: Nickle WR
Agriculture, Universitas Gadjah Mada) for her assistance (ed). Manual of Agricultural Nematology’). Marcel Dekker, New
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Luc M Goodey JB. 1964. Hirschmanniella nom. nov. for Hirschmannia.
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