Control Potentials of Three Entomopathogenic Bacterial Isolates for the Carob Moth, Ectomyelois ceratoniae (Lepidoptera: Pyralidae) in Pomegranates

Ectomyelois ceratoniae (Lepidoptera: Pyralidae) is the primary pest of pomegranates in Saudi Arabia and is mostly controlled using broad-spectrum pesticides. Providing environmentally sound choices to limit reliance on chemical management is a major challenge in the control of E. ceratoniae and, as a consequence, in the protection of pomegranate crops from its invasion. Entomopathogenic bacteria (EPB) symbiotically associated with entomopathogenic nematodes (EPNs) are well-known biocontrol agents of soil-dwelling or aerial pests. The bacterium symbiont (EPB) is the real insect-killing biocontrol agent, while the nematode (EPN) serves as a vector. We wondered whether the EPB vector, which is extremely vulnerable to adverse environmental conditions, like drought, high temperatures, and repellent soil microorganisms, could be omitted. We intended to evaluate the biocontrol potential of directly applied EPB cells and cell-free culture media (CFCM) on the larval instar E. ceratoniae. Xenorhabdus budapestensis DSM 16342 (EMA), X. szentirmaii DSM 16338 (EMC), and Photorhabdus luminescens ssp. laumondi (TT01) strains were used. After three days of exposure, the cells of EMA, EMC, and TT01 strains resulted in 100%, 88%, and 79.3% larval mortality rates, respectively. The applied EMA CFCM resulted in 53.7% larval mortality, indicating the presences of (at least) one extremely strong component produced by EMA. We concluded that the direct application of either the EPB cells or the CFCM must be a prospective alternative biocontrol of E. ceratoniae, especially to protect the important fruit (pomegranate, Punica granatum) cultivars. Especially, newly identified local EPB isolates could be applied as bio-pesticides for integrated management practices or organic pomegranate production.

Identification of Bioactive Plant Volatiles for the Carob Moth by Means of GC-EAD and GC-Orbitrap MS

The aim of this study was to validate a workflow that allows structural identification of plant volatiles that induce a behavioral response in insects. Due to the complexity of plant volatile emissions and the low levels at which these bioactive components tend to occur, gas chromatography-electroantennography (GC-EAD) was applied as the prime differentiator technique, i.e., to indicate particular peaks of interest in the chromatogram. In a next step, the analysis was repeated under identical conditions using GC-Orbitrap mass spectrometry (MS). Combining electron impact (EI) ionization and chemical ionization (CI) with the superior spectral resolution and mass accuracy of the technique enabled straightforward identification of these unknowns, with high confidence in a minute amount of time. Moreover, because of the intrinsic sensitivity of the technique, components that occur at trace amounts but may induce disproportional large behavioral responses are evenly well-identified. We were able to positively identify β-caryophyllene as a bioactive compound in female carob moths. Behavioral attraction was negatively correlated with the amount of β-caryophyllene in host fruits. In an oviposition experiment on filter paper, β-caryophyllene was stimulated in the range of 40–100 ng, while concentrations above 200 ng inhibited oviposition.

A new isolate of Nosema fumiferanae (Microsporidia: Nosematidae) from the date moth Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839 (Lepidoptera: Pyralidae)

In this study, a microsporidian pathogen of the date moth (Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839) also known as the carob moth, is described based on light microscopy, ultrastructural characteristics and comparative molecular analysis. The pathogen infects the gut and hemolymph of A. ceratoniae. All development stages are in direct contact with the host cell cytoplasm. Fresh spores with nuclei arranged in a diplokaryon are oval and measured 3.29 ± 0.23 μm (4.18–3.03 μm, n = 200) in length and 1.91 ± 0.23 μm (2.98–1.66 μm, n = 200) in width. Spores stained with Giemsa's stain measured 3.11 ± 0.31 μm (3.72–2.41 μm, n = 150) in length and 1.76 ± 0.23 μm (2.16–1.25 μm, n = 150) in width. Spores have an isofilar polar filament with 10-12 coils. An 1110 bp long alignment of the current microsporidium showed an SSU rRNA gene difference of only 0.0009, corresponding to >99.91% sequence similarity with Nosema fumiferanae, while RPB1 gene sequences were 98.03% similar within an alignment of 969 bp. All morphological, ultrastructural and molecular features indicate that the microsporidian pathogen of A. ceratoniae is the new isolate of the N. fumiferanae and is named here as Nosema fumiferanae TY61.

Seasonal Population Dynamics, Sampling Distribution, and Fixed-Precision Sequential Sampling of Spectrobates ceratoniae (Lepidoptera: Pyralidae) in Pomegranate Orchards

The carob moth, Spectrobates ceratoniae Zeller, is the most destructive pest of pomegranate groves of Iran. Seasonal population dynamics of the pest was studied in pomegranate orchards of Sirvan, Ilam province, in southwestern Iran for 2 yr (2016/2017). Sampling distribution of the pest larvae on pomegranate fruits was evaluated by Taylor’s power law and Iwao’s patchiness index, and a fixed-precision sequential sampling plan of the pest was developed using Green’s model. The adult population peaked in June. The activity period of the larvae was observed from June to October and peaked in October. Sampling distribution of the larvae on pomegranate fruits was random. Estimated optimum sample sizes ranged from 1 to 44 and 1 to 16 fruits at precision levels of 0.25 and 0.1, respectively.