JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2021-035 e2021-35 | Vol. 53

First record of myriophilus (Poinar, 1986) (: ) from Iran; and its efficacy against two economic forest trees pests, Cydalima perspectalis (Walker, 1859) (Lepidoptera: Crambidae) and Hyphantria cunea (Drury, 1773) (Lepidoptera: Erebidae) in laboratory condition

Reihaneh Gholami Ghavamabad1, Ali Asghar Talebi1,*, Mohammad Mehrabadi1, Mohammad Ebrahim Abstract 2 3, Farashiani and Majid Pedram * The box tree moth (BTM), Cydalima perspectalis and the fall 1Department of Entomology, webworm (FWW), Hyphantria cunea are two invasive pests of Faculty of Agriculture, Tarbiat forest trees that have been recorded from Hyracinan forests in Modares University, P.O. Box north Iran for the first time in 2016 and 2002, respectively. In a 14115-336, Tehran, Iran. search for tentative native entomopathogenic species (EPNs) with potential biocontrol ability against lepidopteran pests 2Research Institute of Forests of forest trees in north Iran, Oscheius myriophilus was isolated and Rangelands, Agricultural by soil-baiting method from forests of Amlash in the east of Gilan Research, Education and Extension province. The Iranian isolate of this species is characterized Organization (AREEO), Tehran, Iran. by 862–1,247 μ m long hermaphrodites having 17–20 μ m long 3Department of Plant Pathology, stoma, vulva at 45.5–53.1% of body length, tail 90–126 μ m long, Faculty of Agriculture, Tarbiat common males with 38–49 μ m long spicules and 583–791 μ m Modares University, P.O. Box long dauer larvae. Compared to the type and other populations, 14115-336, Tehran, Iran. no remarkable differences were observed for this population. The phylogenetic affinities of this isolate with other rhabditid *E-mails: [email protected]; were studied using partial sequences of small, and the [email protected] D2-D3 expansion segments of the large subunit ribosomal DNA This paper was edited by (SSU and D2-D3 LSU rDNA). This is the first record of the species Eyualem Abebe. from Iran. The pathogenicity of Iranian isolate of O. myriophilus was evaluated on the larvae of two important aforementioned forest Received for publication trees pests, BTM and FWW under laboratory conditions. The August 21, 2020. results indicated that the suspension of 500 infective juveniles per ml (IJs/ml) of the nematode was the most effective treatment on fifth instar larvae of BTM and FWW, causing 100 and 95% mortality after 48 h, respectively. The lethal concentration 50 (LC50) values of the nematode were 74.5, 152.7, 99.9, and 197.3 IJs/ml on fifth and fourth instar larvae of BTM, and fifth and fourth instar larvae of FWW, respectively, after 48 h at 25°C and 60% relative humidity. Together, present results corroborated the efficacy of the Iranian isolate of Oscheius myriophilus for biocontrolling of BTM and FWW in laboratory conditions.

Keywords Box tree moth, Entomopathogenic nematode, Fall webworm, Forest pests, Gilan, Pathogenicity, Phylogenetic.

© 2021 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Oscheius myriophilus from Iran and its efficacy against two pests: Ghavamabad et al.

The Hyrcanian (Caspian) forests are natural environ­ (Örley, 1880) is one of the richest groups of free-living ments in the north Iran that are known as the nematodes that are found in all kinds of habitats ecological biodiversity hotspots. So far, 3,234 including decaying bacteria-rich substances such as species belonging to 856 genera and 148 families decaying plants and cadavers or are associated with of vascular plants have been reported from this different , especially the insects (Andrássy, region (Akhani et al., 2010). The box tree moth (BTM), 1983). Sudhaus and Fitch (2001) and Sudhaus and Cydalima perspectalis (Walker, 1859) (Lepidoptera: Schulte (1989) reviewed the systematics, phylogeny, Crambidae), is a native pest in the subtropical regions ecology, and biology of the genera of Rhabditidae. of eastern Asia (Bras et al., 2019). It was detected in Based on a recent analysis by Sudhaus (2011), 38 2007 in southwestern Germany (Billen, 2007) and genera are gathered under the family Rhabditidae, the Netherlands (Muus et al., 2009), and in recent harboring 368 valid species. A pictorial key to the 38 years invaded other European countries (Bras et al., valid genera of Rhabditidae was provided by Scholze 2015, 2016). In Iran, it is an invasive and serious and Sudhaus (2011). The genus Oscheius Andrássy, pest of Buxus L. trees, which was first detected 1976 is delimited by the characters of the lip region in Banafsheh Forest Park, Chalus in 2016; and it is and stoma, tail shape, male posterior body region now distributed all over the Hyrcanian forests in the and bursa, spicules and gubernaculum (Scholze and north Iran (Ahangaran, 2016). The damage caused by Sudhaus, 2011). It belongs to the family Rhabditidae, the larvae of this pest in the foliage, often leads to a has two intragenus groupings, namely Dolichura and complete defoliation of the trees (Wan et al., 2014). Insectivorus (Sudhaus, 1976), the first group being The fall webworm (FWW), Hyphantria cunea (Drury, characterized by having a peloderan bursa, thin tubular 1773) (Lepidoptera: Erebidae) is another invasive pest spicule tips; and the second group is characterized in Hyrcanian forests of Iran. The origin of this moth by a leptoderan or pseudopeloderan bursa, and is North America that has become an invasive pest crochet needle-shaped spicules. Cu­rrently, there in Europe and Asia. In Iran, it was recorded for the are 23 species of Insectivora-group and 16 species first time from Gilan province in 2002 (Rezaei et al., of Dolichura-group (Abolafia and Peña-Santiago, 2004). The FWW is a polyphagous insect that feeds 2019). Some species of Oscheius are necromenic on the leaves of wide range of forest trees, orchards, (Dillman et al., 2012; Sudhaus and Schulte, 1989). cultivated crops, and herbaceous plants (Rezaei In recent studies, evidences have corroborated the et al., 2006; Schowalter and Ring, 2017). According pathogenicity of Oscheius spp. on insects, a behavior to the guide to the biological control of pests in of which mainly correlated with the endosymbiont forests by Food and Agriculture Organization of the bacteria belonging to two genera Serratia Bizio, 1823 United Nations (FAO Forestry) by Kenis et al. (2019), and Enterococcus Thiercelin and Jouhaud, 1903 forest pests can be managed by deployment of (e.g. Oscheius rugaoensis (Zhang et al., 2012), O. resistant or tolerant germplasms and also using chongmingensis (Zhang et al., 2008) (Fu and Liu, chemical, cultural, and biological methods in an 2019; Liu et al., 2012), O. myriophilus (Castro-Ortega integrated pest management (IPM) program. The et al., 2020; Liu et al., 2016), O. microvilli (Zhou et al., first classical biological control projects against forest 2017), O. safricana (Serepa-Dlamini and Gray, 2018) insects using parasitoids and predators was carried and O. basothovii (Lephoto and Gray, 2019) (Lephoto out in Europe, Japan, North Africa, and Asia in 1905 et al., 2015). The bacterial symbiont is the impor­ (Doane and McManus, 1981). In a biological control tant and often overlooked determinant of success of pests using nematodes, the mass production of an entomopathogenic nematode (Labaude and and release of Deladenus siricidicola Bedding, Griffin, 2018).O. myriophilus was described as an 1968 (Neotylenchidae) for biological control of Sirex associate of the garden millipede, Oxidis gracilis noctilio Fabricius, 1793 (Hymenoptera: Siricidae) (Koch) (Diplopoda: Paradoxosomatidae) in California was performed in Australian forests (Bedding by Poinar (1986). It was later isolated from millipedes and Akhurst, 1974). Park et al. (2012) showed that in South Australia by Sudhaus and Schulte (1989), Plutella xylostella (L.) larvae were most susceptible European mole cricket, Gryllotalpa gryllotalpa L. from to blumi (Sudhaus, 1974) showing a 88.0% the Black Sea region of Turkey (Erbaş et al., 2017) rate of mortality. The application technology for use of and a sugar cane crop soil, in Mexico (Castro-Ortega EPNs in biological controlling programs was reviewed et al., 2020). Recent studies by Liu et al. (2016) have by Shapiro-Ilan et al. (2006). Also, application of EPNs shown that O. myriophilus is an entomopathogenic for biocontrol of insect pests at above and below nematode; being associated with the bacterium ground; and their commercialization were reviewed Serratia nematodiphila Zhang et al., 2009. Castro- by Lacey and Georgis (2012). The family Rhabditidae Ortega et al. (2020) isolated the bacterial colonies

2 JOURNAL OF NEMATOLOGY

Serratia marcescens Bizio, 1823 from Oscheius moistened filter papers at 20°C (Nguyen and Hunt, myriophilus, the bacterium which causes a 100% 2007). The male and hermaphrodite nematodes were mortality of the 5th instar larvae of Galleria mellonella obtained by dissecting G. mellonella larvae after 3–4 L. in 1 × 105 concentration. According to them, days of the infection with infective larvae in 1% saline Oscheius myriophilus has the potential to be used (NaCl) solution. The infective larvae were appeared in the future as a biological control agent in Mexico. after one week of infection. Adults and infective larvae Fu and Liu (2019) correlated the pathogenicity of of nematode were fixed and transferred to glycerin dauer larvae of O. chongmingensis with the bacterial for light microscopic studies according to De Grisse genera Bacillus Cohn, 1872, Albidiferax Ramana and (1969). Photographs were taken using an Olympus Sasikala, 2009, Acinetobacter Brisou and Prévot, DP72 digital camera attached to an Olympus BX51 1954 and Rhodococcus Zopf, 1981, isolated from the microscope equipped with differential interference gut of the nematode. There have been few studies on contrast (DIC). Specimens were measured by a identification, ecology and biology ofOscheuis spp. in drawing tube attached to a Nikon Eclipse E600 light Iran. Hassani-Kakhki et al. (2012) reported O. tipulae microscope. The voucher specimens of the nematode (Lam and Webster, 1971) from Iran in the shape of an were deposited in Nematology Laboratory of Tarbiat abstract. Oscheuis rugaoensis was reported from Iran, Modares University, Tehran, Iran. Morphological cha­ isolated from the white grub larvae Polyphylla ads­ racters and morphometric data of the type popu­ persa (Motschulsky, 1853) (Coleopera: Scarabaeidae) lation (Poinar, 1986) and other populations reported (Darsouei et al., 2014). Valizadeh et al. (2017) recovered under the species name (see Table 1) were used for three species O. necromenus (Sudhaus and Schulte, morphological comparisons. 1989), O. onirici (Torrini et al., 2015) and O. tipulae in association with bark beetle galleries in Tehran, Molecular methods and phylogenetic Iran. The application of native EPNs is preferred for analyses more successful and environmental low-risk pur­ poses (Labaude and Griffin, 2018). The recovery of For molecular studies, a single live nematode specimen O. myriophilus in present study from north Iran provi­ was transferred to 20 μ l of TE buffer (10 mM Tris-Cl, ded a chance for examining its potential biocontrol 0.5 mM EDTA, pH 9.0; Qiagen) on a clean slide, heat ability on two devastating forest tree pests dominating killed, photographed and squashed using a clean slide north Iran. cover (Fadakar et al., 2020). Three DNA samples were Thus, the objectives of this study were to (i) prepared in this way. The DNA samples were stored characterize the Iranian isolate of O. myriophilus at −20°C until used in polymerase chain reaction recovered from north Iran based upon traditional and (PCR). The PCR to amplify the partial SSU and D2- molecular criteria, and (ii) assess its biocontrol ability D3 expansion segments of LSU rDNA was carried out against the larvae of two invasive forest pests, BTM in a total volume of 35 μ l (12.1 μ l distilled water, 17.5 μ l and FWW, in laboratory condition. Taq 2X PCR Master Mix, AMPLIQON, Denmark, 1.2 μ l of each primers and 3 μ l DNA template). The partial Materials and methods SSU rDNA was amplified using the forward pri­ mer F22 (5’–TCCAA GGAAGGCAGCAGGC–3’) (Dorris Nematode isolation, culture, and et al., 2002) and reverse primer 1573 R (5’–TAC morphological observation AAAGGGCAGGGACGTAAT–3’) (Mullin et al., 2005). Primers used for the D2–D3 expansion segments of Oscheius myriophilus was collected from forests of LSU rDNA amplification were forward primer KK28S- Amlash (37°08’N, 50°16’E), Gilan province, north Iran, 1D1F (5’–AAGGATTCCCTTAGTAACGGCGAGTG–3’) in May 2019 using the baiting technique (Bedding and (Kiontke et al., 2004), reverse primer D3B (5’–TCGG Akhurst, 1975) and the last instar larvae of Galleria AAGGAACCAGCTACTA–3’) (Nunn, 1992). The thermal mellonella. No other nematodes were collected using cycling program for amplification was as follows: this method in this experiment. The dead insect larvae denaturation at 95°C for 4 min, followed by 35 cycles were transferred to Petri dishes with filter paper and of denaturation at 94°C for 30 s, annealing at 52°C for covered with plastic according to white trap method 40 s, and extension at 72°C for 80 s. A final extension (White, 1927). Dauer/infective larvae were emerged was performed at 72°C for 10 min. To check the and stored in distilled water at 8°C (Kaya and Stock, successful amplification, 2 μ l of the PCR product was 1997). In all, 20 last instar larvae of G. mellonella loaded on agarose gel including DNA Green Viewer™. were exposed to 2,000 infective juveniles of the The PCR products were purified and sequenced by nematode in a Petri dish (60 × 15 mm) plates lined with the Macrogen Macrogen Corporation, South Korea.

3 Oscheius myriophilus from Iran and its efficacy against two pests: Ghavamabad et al.

– – – – – – – – – larvae 25 ± 2.2 4.7 ± 0.2 9.3 ± 0.9 Turkey (4.3–5.2) 25.4 ± 2.7 (8.3–11.6) Erbaş et Infective 134.6 ± 3.2 al. (2017) (20.5–28.5) 630.2 ± 31.5 Gryllotalpa gryllotalpa (571.3–693.9) (128.8–139.8) – – – – – – – – – – – – – – (3–4) < 4.5 (18–21) (4.2–8.5) (8.9–13.0) (792–1,530) Hermaphrodite (1989) Australia O. gracilis – – – – – – – – – – – – – – – – – Sudhaus and Schulte Male (4.0–6.1) (14.4–21.8) (841–1,175) – – – – – – – – – – – – – – 6 23 564 129 larvae (504–611) (120–136) Infective

– – – – – – 10 20 62 3.2 185 1,320 (18–21) (1986) (6.8–7.7) (2.2–3.5) (174–193) (19.1–21.3) California (1,200–1,500)

Oxidis gracilis Hermaphrodite – – – – – – 10 17 63 3.2 187 1,270 Male (16–19) (161–200) (18.4–21.9) (5.16–7.36) (14.8–20.4) Type population described by Poinar (830–1,470) (Poinar, 1986) recovered from Gilan province, north Iran, and – – 20 (14–20) larvae 4.9 ± 0.3 7.1 ± 0.4 5.5 ± 0.3 2.7 ± 0.3 (4.6–5.7) (6.6–7.9) (5.1–6.6) (2.5–4.0) 24.2 ± 1.8 16.1 ± 1.4 27.8 ± 3.4 (583–791) (125–145) Infective 134.5 ± 7.1 (21.0–27.9) 672.3 ± 71.4 20 Iran (4–4.5) (17–20) 5.8 ± 0.5 9.4 ± 0.9 5.0 ± 0.5 4.2 ± 0.2 (5.0–6.9) (4.1–5.7) 19.6 ± 2.1 49.9 ± 2.0 17.6 ± 0.8 52.3 ± 6.8 (8.0–10.9) (144–200) (17.0–22.7) (45.5–53.1) (862–1,247) 176.3 ± 17.1 1,020 ± 129.6 This study Hermaphrodite Oscheius myriophilus

Galleria mellonella – – 20 (3–4) Male (17–18) 5.1 ± 0.2 3.4 ± 0.5 (4.8–5.5) (1.5–1.8) 196 ± 9.7 15.3 ± 2.1 18.1 ± 0.9 1.7 ± 0.07 17.5 ± 0.5 (180–208) 66.8 ± 11.5 (13.2–18.7) (16.6–19.2) (867–1,053) 1,002.1 ± 61.3 Locality Host insect Character n L a b c c' V% = (distance from anterior end to vulva/L) × 100 Stoma length Stoma width Pharynx length Max. body diam. Table 1. Morphometrics of comparison with data of the type population, population from Australia and Turkey.

4 JOURNAL OF NEMATOLOGY – – – – – – – – – – – – – – – – – – – – 12.3 ± 1 108 ± 6.7 82.1 ± 6.2 110.5 ± 4.9 (10.4–13.8) (72.2–92.2) (21.0–30.4) (100–116.8) (97.8–118.8) – – – – – – – – – – – – – – – – – – – – – – – – – 32 1.5 (1.4–2.3) (52–108) – – – – – – – – – – – – – – – – – – – – – – – – – – – (27–39) (52–72) – – – – – – – – – – – – – – – – – – – – 89 15 78 107 (14–16) (75–80) (19–26) (83–96) (97–114) – – – – – – – – – – – – – – – – – – 79 25 117 179 139 (56–96) (22–28) (57–70) (108–135) (165–190) (126–146) – – – – – – – – – – – – 39 65 47 10 28 198 150 0.87 (8–11) (32–45) (56–72) (32–54) (19–32) (38–80) (149–229) (117–165) (0.64–1.20) – – – – – – – – – – – – – – – – (15–18) (24–33) (85–100) (83–105) 94.5 ± 6.7 16.9 ± 0.9 93.7 ± 5.0 87.1 ± 3.5 (110–129) 125.5 ± 5.0 116.3 ± 4.6 (82.5–94.4) (118.0–136.3) – – – – – – – – – – – – (55–80) (17–26) (42–66) 1.7 ± 0.1 (90–126) (1.1–1.8) 63.7 ± 4.8 21.7 ± 2.4 85.1 ± 6.8 (135–167) (105–135) (76.3–100) 108.1 ± 9.8 149.3 ± 9.3 121.1 ± 9.7 138.7 ± 10.2 (124.3–160.0) – – – – (7–8) (30–35) (52–60) (38–49) (18–21) (47–79) 7.1 ± 0.3 (0.8–1.1) 126 ± 4.7 32.3 ± 1.8 55.3 ± 3.3 44.5 ± 3.8 20.0 ± 1.0 0.9 ± 0.09 84.6 ± 3.7 45.1 ± 2.5 (273–323) (142–177) (118–130) 137.8 ± 8.2 (78.8–90.5) (41.6–48.8) 300.2 ± 16.8 165.9 ± 11.1 (126.6–150.0) = (E pore from Rectum length Anal body diam. Tail Ratio of tail length to rectum Spicules length Spicules width Gubernaculum length Gubernaculum width D% anterior end/Pharynx length) × 100 E% = (E pore from anterior end/Tail) × 100 SW% = (Spicules length/Anal body diam.) × 100 GS% = (Gubernaculum length/Spicules length) × 100 E pore from anterior end Nerve ring from anterior end

5 Oscheius myriophilus from Iran and its efficacy against two pests: Ghavamabad et al.

The newly generated DNA sequences of Oscheius July 2019. They were reared on mulberry leaves under myriophilus were compared with those of other laboratory conditions at growth chambers (24°C, 60% nematode species available in GenBank using BLAST. RH, 16:8 h L:D) in plastic containers. Mulberry leaves The maximal number of sequences of Oscheius spp. were changed daily, not allowing to dry completely. The (Abolafia and Peña-Santiago, 2019) and sequences susceptibilities of fourth and fifth instar larvae of BTM from several other classic rhabditid genera were and FWW to the Oscheius myriophilus were evaluated selected for reconstructing of the phylogenetic trees. under laboratory conditions. The larvae were exposed The alignment of SSU sequences was done using the to dauer stage of the nematode in 24-well plates (SPL Q-INS‐i algorithm of online version of MAFFT version Life Sciences Co., Ltd. Korea) having a small hole in the 7 (http://mafft.cbrc.jp/ alignment/server/) (Katoh and lid, including a piece of Whatman filter paper and small Standley, 2013). To eliminate the ambiguously aligned pieces of fresh washed leaves. A single larva of insect parts, the online version of Gblocks 0.91b with all pests was placed in each well, and inoculated with the the three less stringent parameters was used (http:// infective juveniles of the nematode. Each larva in the molevol.cmima.csic.es/castresana/Gblocks_server. treatment was inoculated with 0, 10, 25, 50, 100, 200, html). The alignment of LSU rDNA D2-D3 sequences 300, and 500 IJs per 1 ml of distilled water. The control was done using ClustalW and t