Can Symbiotic Bacteria (Xenorhabdus and Photorhabdus) Be More Efficient than Their Entomopathogenic Nematodes against Pieris rapae and Pentodon algerinus Larvae?

Pieris rapae and Pentodon algerinus are considered a global threat to agricultural crops and food security; hence, their control is a critical issue. Heterorhabditid and Steinernematid nematodes, along with their symbiotic bacteria, can achieve the optimal biocontrol agent criterion. Therefore, this study aimed to evaluate the efficacy of Heterorhabditis bacteriophora, Steinernema riobravis, and their symbiotic bacteria (Xenorhabdus and Photorhabdus) against P. rapae and P. algerinus larvae. The virulence of entomopathogenic nematodes (EPNs) was determined at different infective juvenile concentrations and exposure times, while the symbiotic bacteria were applied at the concentration of 3 × 107 colony-forming units (CFU)/mL at different exposure times. Gas chromatography–mass spectrophotometry (GC-MS) analysis and the cytotoxic effect of Photorhabdus sp. and Xenorhabdus sp. were determined. The results indicated that H. bacteriophora, S. riobravis, and their symbiotic bacteria significantly (p ≤ 0.001) induced mortality in both insect species. However, H. bacteriophora and its symbiont, Photorhabdus sp., were more virulent. Moreover, the data clarified that both symbiotic bacteria outperformed EPNs against P. rapae but the opposite was true for P. algerinus. GC-MS analysis revealed the main active compounds that have insecticidal activity. However, the results revealed that there was no significant cytotoxic effect. In conclusion, H. bacteriophora, S. riobravis, and their symbiotic bacteria can be an optimal option for bio-controlling both insect species. Furthermore, both symbiotic bacteria can be utilized independently on EPNs for the management of both pests, and, hence, they can be safely incorporated into biocontrol programs and tested against other insect pests.

Survey and identification of entomopathogenic nematodes in the province of Cotabato, Philippines, for biocontrol potential against the tobacco cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Background The tobacco cutworm, Spodoptera litura [Fab.] (Lepidoptera: Noctuidae), is a devastating insect pest of several crops. Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are used as an alternative control measure in lieu of the hazardous synthetic chemical applications. Results A survey of naturally occurring EPNs was conducted across the province of Cotabato, Philippines, covering a total of 5 municipalities with 25 villages. Galleria-baiting technique was employed to recover nematodes from peanut and grassland soils. Out of 50 soil samples collected, only 5 samples harbored nematodes, indicating a recovery of 10%. Preliminary morphological data identified only one EPN under the genera Heterorhabditis (1 isolate), whereas 4 were facultative necromenic nematodes from the genera Metarhabditis (2 isolates) and Oscheius (2 isolates). Analysis of D2D3 segments of the 28S rDNA confirmed high sequence similarity to Heterorhabditis indica, Metarhabditis rainai, Oscheius insectivora, and Oscheius sp. This is the first record of H. indica and M. rainai in the entire region, whereas the first record for Oscheius spp. in the Philippines. Furthermore, the biocontrol potential of the local H. indica infective juvenile (IJ) populations (PIGCD1) isolated from peanut was assessed against the tobacco cutworm, S. litura, under laboratory conditions. The mean percentage mortality caused by H. indica on S. litura at 7 different concentrations ranged from 0-100% at 24 h post inoculation. The lethal concentration (LC50) required to kill 50% of the S. litura larvae population with H. indica was 7.13±1 (IJs/larva). Conclusions The use of Galleria-baiting method is a convenient approach to detect EPNs including other facultative necromenic nematodes from the soils. Obtained data indicated that the local H. indica isolate can be a promising alternative measure to suppress the economically important insect pest, S. litura, and this may provide significant outlook to establish the biocontrol program in the country.

Keefektifan Patogenesitas Steirmema Carpocapsae (all strain) terhadap Hama Plutella xylostella L.

The utilization of entomopathogenic nematodes is an example of the uses of an environmentally friendly biological agent. Entomopathogenic nematodes of the family Steinernematidae and Hetrorhabditidae are very potential to control insect pests. The purpose of this study was to study the pathogenicity of the entomopathogenic nematode Steinernema carpocapsae (all strains) as a biological control against Plutella xylostella. This research includes 2 stages, namely the preparation stage and laboratory experiments. The experimental design in this study was a completely randomized design (CRD) consisting of six treatments and three replications. The treatments tested were entomopathogenic nematode concentrations consisting of six levels: 0, 8, 16, 32, 64 and 128 infective juvenile per ml. The LC50 and LT50 values were calculated using Probit analysis. The results of observations of nematodes that enter the insect body and pest mortality were analyzed using analysis of variance (ANOVA), once showing a significant difference, it was then continued to the DMRT test at 5% level. Based on the results of the study, the highest pathogenicity against Plutella xylostella was Steinernema carpocapsae (all strains) when compared to Steinernema glaseri (NC) and Steinernema sp. local isolates. Resistance to Steinernema carpocapsae (all strains) and the LC50 value were determined by the age of Plutella xylostella larvae. The bigger and older the larvae, the more its resistance to Steinernema carpocapsae (All strains) and the LC50 value. The number of entomopathogenic nematodes that enter the body of Plutella xylostella increased with increasing length of contact time.Penggunaan nematoda entomopatogen merupakan salah satu pemanfaatan agensia hayati yang ramah lingkungan. Nematoda entomopatogen dari famili Steinernematidae dan Hetrorhabditidae sangat potensial untuk mengendalikan serangga hama. Tujuan dari penelitian ini adalah mempelajari patogenisitas nematoda entomopatogen Steinernema carpocapsae (all strain) sebagai pengendali hayati terhadap hama Plutella xylostella. Penelitian ini meliputi 2 tahap yaitu tahap persiapan dan percobaan laboratorium. Rancangan percobaan pada penelitian ini adalah Rancangan Acak Lengkap (RAL) yang terdiri enam perlakuan dan tiga ulangan. Perlakuan yang diujikan adalah konsentrasi nematoda entomopatogen yang terdiri enam taraf: 0, 8, 16, 32, 64 dan 128 infektif juvenile/ml. Nilai LC50 dan LT50 dihitung menggunakan analisis probit. Hasil pengamatan nematoda yang masuk dalam tubuh serangga dan mortalitas hama dianalisis menggunakan analisis sidik ragam (ANOVA), apabila menunjukkan beda nyata dilanjutkan uji DMRT taraf 5%. Berdasarkan hasil penelitian, patogenisitas tertinggi terhadap Plutella xylostella adalah Steinernema carpocapsae (all strain) bila dibandingkan dengan Steinernema glaseri (NC) dan Steinernema sp. isolat lokal. Ketahanan terhadap Steinernema carpocapsae (All strain) dan nilai LC50 ditentukan umur larva Plutella xylostella. Semakin besar dan tua umur larva, ketahanan terhadap Steinernema carpocapsae (All strain) dan nilai LC50 juga semakin meningkat. Jumlah nematoda entomopatogen yang masuk kedalam tubuh Plutella xylostella semakin banyak seiring dengan bertambah lamanya waktu kontak.

Morphology, molecular profiles and distribution of Japanese populations of Steinernema tielingense Ma, Chen, Li, Han, Khatri-Chhetri, De Clercq & Moens, 2012 (Rhabditida: Steinernematidae)

Summary Steinernema tielingense was found to be widely distributed in Japan. The molecular characteristics of the nematodes isolated from Japan are basically in accordance with those mentioned in the original description of S. tielingense. However, the Japanese isolates are morphologically distinct from the type population by the following characters: length of the infective juvenile, 711 (671-769) μm, excretory pore of adult males and infective juveniles located posterior mid-pharynx, D% < 50%, smaller H value of 35% and shorter spicules in adult males of both generations. Although discrepancies in morphology and morphometric data between the Chinese isolate and Japanese isolate were observed, the molecular data matched and on this basis the Japanese isolate was identified as being conspecific with S. tielingense. The symbiotic bacterium isolated from the Japanese population of S. tielingense was identified as Xenorhabdus bovienii.

Surface culture of Steinernema sp. on two solid media and their pathogenicity against Galleria mellonella

The use of native entomopathogenic nematodes as biocontrol agents is a strategy to decrease the environmental impact of insecticides and achieve sustainable agriculture crops. In this study, the effect of the surface culture of Steinernema sp. JAP1 over two solid media at 23–27°C on infective juvenile (IJ) production and pathogenicity against Galleria mellonella larvae were investigated. First, the bacterial lawn on the surface of the media with egg yolk (P2) or chicken liver (Cl) were incubated in darkness at 30°C for 48 and 72 h, and 100 surface-sterilized IJs were added. Four harvests were conducted within the next 35 days and the mean accumulated production was superior on Cl (210 × 103 IJs) than on P2 (135 × 103 IJs), but the productivity decreased up to 10% when the incubation time of the bacterial lawn was of 72 h. The mean pathogenicity of in vitro- and in vivo-produced IJs were of 47–64% and 31%, respectively. It is worth noting that none of the two solid media had a statistically significant difference in IJ pathogenicity. Considering that the maximum multiplication factor of IJs on solid media was 2108 and that the pathogenicity against G. mellonella was outstanding, Steinernema sp. has a good potential for in vitro mass production.

Effect of agitation speed on the density of bacteria Photorhabdus luminescens and the population dynamics of nematodes Heterorhabditis megidis in liquid culture

Liquid culture is the most scalable technology for the industrial production of entomopathogenic nematodes. Variability of the recovery after inoculation into cultures of Photorhabdus luminescens remains a persistent problem in the mass production of Heterorhabditis sp. In order to enhance infective juvenile (IJ) recovery and improve nematode population management, we analysed the correlation between the nematode Heterorhabditis megidis (strain KV – 136) development in liquid cultures, the density of bacteria of P. luminescens and the culture agitation speed. Analyses focused on the impact of different agitation speeds (160 rpm and 200 rpm) on the dynamics of population growth of H. megidis in liquid cultures at constant biotic and abiotic parameters (initial dose of nematodes introduced to the culture 2300 IJs/ml, temperature 25°C, the number of bacterial colonies 0.3 × 107/ml). The performed experiments showed that the agitation speed of 200 rpm favourably affected the density of bacteria of P. luminescens (24.14 × 107/ml). High density of bacteria at this agitation speed resulted in an earlier (on the fifth day of the culture) maximum increase in the number of hermaphroditic individuals (1239.6 H/ml) than in the culture at an agitation speed of 160 rpm.

Morphological, Molecular and Ecological Characterization of a Native Isolate of Steinernema Feltiae (Rhabditida: Steinernematidae) From Southern Chile

Background: Steinernema feltiae is an entomopathogenic nematode used in biological control programs with a global distribution. It has been shown that populations of S. feltiae may have phenotypic plasticity derived from local adaptation and may vary in different traits, such as location and penetration of the host. This is the first time that a Chilean isolate has been described in detail, taking into account morphological, molecular and ecological characteristics. Methods: S. fealtiae was detected a few years ago in southern regions of Chile in the town Lican Ray and was morphologically and morphometrically described, along with a molecular description based on the ITS and D2-D3 regions. Some ecological characteristics were determined, including the temperature requirements for completion of its life cycle and the effect of three water levels in soil for optimal reproduction. Results: Morphometric characteristics of different life stages show large intraspecific variability in averages and ranks compared with isolates from different geographical origins. The molecular data also show intraspecific variability with respect to other isolates. The lower, optimal and higher temperatures found to limit the infestation and reproduction of Galleria mellonella were 10, 20 and 30 °C respectively, and emergence from the host larvae occurred approximately ten days after inoculation. Differences were observed in offspring emerging from the host, and the 120 infective juveniles (IJ)/larvae dose was the most prolific at 20 °C. The water content of the soil did not affect the number of infective juvenile invaders, penetration efficacy (%), and time of emergence of the IJ or offspring per larvae, but it caused a delay in achieving full mortality at the permanent wilting point with respect to saturation and field capacity.Conclusions: Data obtained in this study are valuable since they outline some environmental requirements of this strain to perform optimally in the event of being used as a soil pest bioantagonist.

Phasmarhabditis kenyaensis n. sp. (Nematoda: Rhabditidae) from the slug, Polytoxon robustum, in Kenya

Summary A new species of Phasmarhabditis was isolated from the slug, Polytoxon robustum, from Nairobi, Kenya. The nematode was identified using morphological, morphometric, molecular and phylogenetic analyses. Phasmarhabditis kenyaensis n. sp. is characterised by an infective juvenile with the longest body length in the genus, measuring 1232 (1107-1336) μm, by the presence of males with a bursa bearing nine bilateral pairs of genital bursal papillae and one pair of papilliform phasmids flanking the tail, cephalate paired spicules, with an arc length of 71 (57-81) μm, as well as by females with a vulva located at the mid-body region and a conoid tail shape, with two phasmids located at ca 40% of the tail length. The molecular phylogeny of the new species, as inferred from its SSU (small subunit) rRNA gene, places P. kenyaensis n. sp. genetically close to undescribed phasmarhabditids from South Africa, suggesting an African grouping, while the D2-D3 (large ribosomal subunit) and ITS region analyses relate P. kenyaensis n. sp. to P. meridionalis, with weak bootstrap support. This is the third new Phasmarhabditis species described from the African continent, the new species bringing the total known complement of the genus to 14 species. A morphometric compendium to all species cultured in vivo is supplied.

Steinernema riojaense n. sp., a new entomopathogenic nematode (Nematoda: Steinernematidae) from Spain

Summary A new species of entomopathogenic nematode, Steinernema riojaense n. sp., was isolated during a survey in La Rioja province of Spain. Morphological and morphometric traits place the new species in the glaseri-group, which is characterised by having the largest infective juvenile (IJ) with an average body length in excess of 1000 μm. The IJ of S. riojaense n. sp. possess a body length of 1043 (869-1172) μm, the lateral fields are formed by eight equally spaced and developed ridges (nine incisures) in the mid-body region, giving the formula 2, 6, 8, 4, the excretory pore is located posterior to the mid-pharynx region (D% = 58), and the hyaline region occupies ca half of the tail length. The male spicules are moderately curved with a sharp tip and are golden-brown in colour with the manubrium elongate and having a length to width ratio of 1.7:1. The first-generation male tail is non-mucronate, whereas a small papilla-like mucron is developed in the second generation. There are 11 pairs of genital papillae plus a single precloacal papilla. The first-generation female possesses a moderately protruding vulva and a slightly protruding postanal swelling. The second-generation female possesses a slightly protruding vulva and a postanal swelling. The new species is further characterised by sequences of the internal transcribed spacer (ITS) and partial 28S regions (D2-D3) of the ribosomal DNA (rDNA). Phylogenetic analyses confirm that S. riojaense n. sp. belongs to the glaseri-group and forms a monophyletic subclade with other European species.

VIABILITAS DAN EFEKTIVITAS FORMULA NEMATODA Steinernema sp. TERHADAP HAMA PENGGEREK BUAH KAPAS Helicoverpa armigera HUBNER

<p>ABSTRAK<br />Entomopatogen dari genus Steinernema berpotensi digunakan<br />sebagai pengendali berbagai serangga hama, terutama ordo Lepidoptera,<br />seperti Helicoverpa armigera. Penggunaan Steinernema untuk<br />pengendalian H. armigera akan menguntungkan karena aman terhadap<br />lingkungan, mudah diproduksi massal, toleran terhadap berbagai macam<br />pestisida, dapat aktif mencari serangga sasaran, tidak menyebabkan<br />resisten dan resurjensi, serta dapat diaplikasikan dengan alat semprot<br />standar. Namun, formula pestisida hayati mengandung Steinernema masih<br />sangat terbatas. Tujuan penelitian adalah membuat formula Steinernema<br />sp. yang efektif terhadap hama penggerek buah kapas (H. armigera).<br />Penelitian dilaksanakan di laboratorium Patologi Serangga, Balai<br />Penelitian Tanaman Pemanis dan Serat, Malang mulai bulan Mei-Juli<br />2010. Larva instar III Steinernema sp. dibuat dalam 6 macam formula<br />perlakuan dengan bahan pembawa (carrier) berbeda-beda, yaitu (1)<br />suspensi (akuades + sukrosa), (2) pellet-2 (sekam padi), (3) pellet-1<br />(tanah liat + arang), (4) agar + spon, (5) kapsul (Ca-alginat), dan (6)<br />kontrol (akuades). Setiap formula diinokulasikan 10 6 juvenil infektif (JI).<br />Masing-masing perlakuan disusun dalam rancangan acak lengkap (RAL)<br />dengan tiga kali ulangan. Penurunan jumlah juvenil infektif (JI) pada<br />setiap formula yang diamati per minggu selama ± 4 minggu. Isolat yang<br />digunakan untuk penelitian ini berasal dari Asembagus. Untuk<br />mengevaluasi efektivitas formula, larva H. armigera diperlakukan dengan<br />JI yang berasal dari masing-masing formula setiap minggu selama empat<br />minggu. Perlakuan ini menggunakan rancangan acak lengkap dengan<br />empat ulangan. Jumlah penurunan JI setiap minggu selama empat minggu<br />setelah perlakuan. Persentase JI yang hidup pada pellet-1, suspensi, pellet-<br />2, agar + spon, kapsul, dan kontrol berturut-turut sebesar 53; 12,4; 44;<br />63,8; 17,6; dan 5%. Pada minggu pertama sampai minggu keempat setelah<br />perlakuan terlihat bahwa formula yang paling baik mempertahankan JI<br />adalah agar + spon dan kemudian berturut-turut diikuti oleh pellet-1,<br />pellet-2, kapsul, suspensi. Steinernema sp. yang disimpan selama empat<br />minggu dalam berbagai bentuk formula terhadap H. armigera berkisar<br />antara 80-99%. Formula agar dan spon paling baik untuk menyimpan<br />Steinernema sp. selama empat minggu, karena formula ini memberikan<br />tingkat viabilitas dan efektivitas Steinernema sp. paling tinggi.<br />Kata kunci : efektivitas, formula, Helicoverpa armigera, Steinernema sp.,<br />viabilitas</p><p>ABSTRACT<br />Entomopathogenic nematodes genus Steinernema for are potential<br />to be used as a control for various insect pests, especially ordo<br />Lepidoptera, such as Helicoverpa armigera. The use of Steinernema to<br />control H. armigera is beneficial because it is environmentally friendly,<br />easy to produce, tolerant to several pesticides, actively search the target<br />insect, does not cause resistance and resurgence, and can be applied by<br />using standard sprayer. Unfortunately, biological pesticide containing<br />Steinernema is still limited. The study was aimed at formulating a<br />biological agen containing Steinernema sp. to control the cotton bollworm<br />weevil (H. armigera). The experiment was conducted at insect pathology<br />laboratory, Indonesian Sweetener and Fiber Crops Research Institute,<br />Malang from May to July 2010. Steinernema sp. instar III larvae was<br />formulated in six different forms such as Pellet-1 (clay+carbon),<br />suspension (distilled water+sucrose), Pellet-2 (rice husk), agar+sponge,<br />capsule (Ca-alginate), and control (distilled water). Each formulation was<br />inoculated with 10 6 Infective Juvenile (IJ). Each treatment was arranged in<br />completely randomized design (CRD) with three replicates. Each<br />formulation observed showed decrease in IJ number every week for + 4<br />weeks. Isolates used for this research were originated from Asembagus<br />experimental station. For evaluation of the formulation effectiveness, H.<br />armigera larvae was treated using IJ from the formula weekly for four<br />weeks. On the first week after treatment, the percentages of living IJ in<br />Pellet-1, suspension, Pellet-2, agar+sponge, capsule, and control were 53;<br />12.4; 44; 63.8; 17.6; and 5%, respectively. Effectiveness of Steinernema<br />sp. stored for four weeks in various formulations against H. armigera<br />ranged from 80-99%. The best formula of Steinernema sp for storage was<br />agar+sponge because of its ability to viability and effectiveness of<br />Steinernema sp.<br />Key words: effectiveness, formulation, Helicoverpa armigera, viability,<br />Steinernema sp.,</p>