Isolation, Identification, and Biocontrol Potential of Entomopathogenic Nematodes and Associated Bacteria against Virachola livia (Lepidoptera: Lycaenidae) and Ectomyelois ceratoniae (Lepidoptera: Pyralidae).

Saqer S Alotaibi,Sarah Alharthi,Mohd Asif,Rania H Wahdan,Hadeer Darwish,Amal Al-Barty,Ahmed Noureldeen,Madiha Zaynab,Akram Alghamdi,Alaa Baazeem

doi:10.3390/biology11020295

Abstract

Simple SummaryFor sustainable agriculture, there is a need to identify and evaluate more biocontrol agents, including entomopathogenic nematodes (EPNs). In this study, EPNs and their associated entomopathogenic bacteria symbionts (EPBs) were isolated and identified from 320 soil samples collected in Taif, Saudi Arabia. The biocontrol efficacy of EPNs and EPBs was also evaluated against third instar larvae of the pomegranate butterfly, Virachola livia, and the carob moth, Ectomyelois ceratoniae, two important insect pests of pomegranate, under laboratory conditions. Our results show that the EPNs Steinernema spp. were more virulent than Heterorhabditis spp. against the two pomegranate insects. In addition, the EPB Stenotrophomonas maltophilia CQ1, isolated from Steinernema spp., surpassed Pseudomonas mosselii SJ10, associated with Heterorhabditis spp., in their ability to kill V. livia or E. ceratoniae larvae. We conclude that either application of EPNs’ infective juveniles (IJs) or their associated EPBs could serve as potential biocontrol agents for V. livia and E. ceratoniae.Virachola livia (Lepidoptera: Lycaenidae) and Ectomyelois ceratoniae (Lepidoptera: Pyralidae) are the key pests of pomegranates in Saudi Arabia that are managed mainly using broad-spectrum pesticides. Interactions between the entomopathogenic nematodes (EPNs) Steinernematids, and Heterorhabditids, and their entomopathogenic bacterial symbionts (EPBs) have long been considered monoxenic 2-partner associations responsible for killing insects and, therefore, are widely used in insect pest biocontrol. However, there are limited reports identifying such organisms in Taif, Saudi Arabia. The current study aimed to identify the EPNs and their associated bacteria isolated from Taif, Saudi Arabia, and evaluate their biocontrol potential on third instar larvae of V. livia and E. ceratoniae under laboratory conditions. A total of 35 EPN isolates belonging to Steinernema (20) and Heterorhabditis (15) were recovered from 320 soil samples. Twenty-six isolates of symbiotic or associated bacteria were isolated from EPNs and molecularly identified as Xenorhabdus (6 isolates), Photorhabdus (4 isolates), Pseudomonas (7), or Stenotrophomonas (9). A pathogenicity assay revealed that Steinernema spp. were more virulent than Heterorhabditis spp. against the two pomegranate insects, with LC50 values of 18.5 and 13.6 infective juveniles (IJs)/larva of V. livia for Steinernema spp. and 52 and 32.4 IJs/larva of V. livia for Heterorhabditis spp. at 48 and 72 h post-treatment, respectively. Moreover, LC50 values of 9 and 6.6 IJs/larva (Steinernema spp.) and 34.4 and 26.6 IJs/larva (Heterorhabditis spp.) were recorded for E. ceratoniae larvae at 48 and 72 h post-treatment. In addition, the EPB Stenotrophomonas maltophilia CQ1, isolated from Steinernema spp., surpassed Pseudomonas mosselii SJ10, associated with Heterorhabditis spp., in their ability to kill V. livia or E. ceratoniae larvae within 6 h post-application, resulting in 100% mortality in both insects after 24 and 48 h of exposure. We conclude that either application of EPNs’ IJs or their associated EPBs could serve as potential biocontrol agents for V. livia and E. ceratoniae.

Highlights

Nematodes that parasitise insects, known as entomopathogenic nematodes (EPNs), have been identified within 23 nematode families [1]
Following methods by Alotaibi et al [48], pomegranate butterfly (V. livia) and carob moth (E. ceratoniae) larvae were collected from commercial pomegranate orchards located in Taif, Saudi Arabia, and reared on a wheat bran diet (300 g of wheat bran, 50 g of sugar, 120 mL of water, 130 mL of glycerol, and 9 g of yeast), whereas adults were fed with a 10% honey–water solution
Our findings are in accordance with those reported by Noureldeen [25], who recorded the occurrence of one EPN species, Steinernema sp., in the rhizosphere of pomegranate trees in Taif, Saudi Arabia

Summary

Introduction

Known as entomopathogenic nematodes (EPNs), have been identified within 23 nematode families [1]. Photorhabdus and Xenorhabdus are Gram-negative bacteria of the family Enterobacteriaceae and are symbiotically associated with the entomopathogenic nematodes Heterorhabditis and Steinernema, respectively [3]. The infective juveniles (IJs) of Steinernematid and Heterorhabditid nematodes carrying symbiotic bacteria in their midguts live in the soil of diverse ecological systems [4]. These nematodes actively seek insect hosts, penetrating through an insect’s mouth, anus, or spiracles to reach the hemocoel, where symbiotic bacteria (e.g., Xenorhabdus spp. and Photorhabdus spp.) are released [5]. The symbiotic bacteria replicate rapidly and cause septicaemia in insects [8]. This process converts the insect cadaver into suitable food for nematode development and reproduction

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