Stability of entomopathogenic bacteria, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis> and <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>, during in vitro culture

The entomopathogenic nematode–bacteria complexes Heterorhabditis bacteriophora/Photorhabdus luminescens and Steinernema carpocapsae/Xenorhabdus nematophila are mass produced for use as biological insecticides. Stability of the bacterial partner in culture is essential for maintaining traits important for both biological control and production. Two geographically distinct strains of each bacterial species were isolated from their nematode partners and serially subcultured on in vitro media to assess trait stability. Subculturing resulted in a shift to secondary cell production in one P. luminescens strain and both X. nematophila strains within ten in vitro culture cycles. However, when cell phenotypic variation was controlled in X. nematophila strains by regular selection for primary variants, no trait change was detected in the primary variant after prolonged subculture. When P. luminescens cell phenotypic variation was controlled by selection for primary variants, changes in the primary variant of both strains were noted including reductions in cell and inclusion body size and inclusion body prevalence. Bacterial ability to cause lethal infections following injection into the hemocoel of Tenebrio molitor larvae declined by more than half in primary variants of one P. luminescens strain. Conversely, yield was enhanced, with the subcultured P. luminescens strains showing 53.5 and 75.8% increases in primary cell density. Field adapted traits of primary variant P. luminescens strains tend to deteriorate during in vitro culture as tradeoffs for gains in yield. In vitro producers of the P. luminescens/H. bacteriophora complex must weigh the need for superior bacterial yield against the need to preserve traits important for biological control.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 191–197.Original Russian Text Copyright © 2005 by Deryabin, Polyakov.     

Nutritive significance of crystalline inclusion proteins of Photorhabdus luminescens in Steinernema nematodes

Phase I cells of Photorhabdus luminescens produce two types of intracellular crystalline inclusion proteins designated CipA and CipB. The genes encoding CipA and CipB proteins from P. luminescens H06 were expressed respectively in Escherichia coli and these cells were used to feed the axenic first juveniles (J1) of three Steinernema nematode isolates in liquid cultures and on agar plates. In liquid cultures, the axenic J1 juveniles of all three test Steinernema nematode isolates were able to produce next dauer juveniles (DJs) in the E. coli cultures with at least one of the expressed Cip proteins, but unable to develop beyond the next J1 stage without expressed Cip proteins. For each target nematode isolate, addition of the supernatant of the bacterial culture of its Xenorhabdus symbiont to the tested liquid cultures did not induce the formation of DJs. However, on LB agar plates with different test E. coli cultures, all J1 juveniles of the three Steinernema strains finally developed into next DJs. It seemed that the metabolite pathway of the test bacteria in both culture systems was different. The presence of the Cip proteins has a significant influence on the DJ formation of the Steinernema nematodes in liquid culture system.     

荧光发光杆菌TT01品系pirA2B2 基因的克隆表达与杀虫活性

【目的】Photorhabdus luminescens TT01基因组中的一对ORF plu4437-plu4436(简称pirA2B2)的预测氨基酸序列与另一对已证明编码产物有口服杀虫活性的ORF plu4093-plu4092(简称pirA1B1)有50%和45%的一致性,本文旨在研究pirA2B2基因座的表达产物是否也有杀虫活性。【方法】PCR扩增并克隆了pirA2,pirB2和pirA2B2基因,构建了重组表达载体pQE-pirA2,pQE-pirB2和pQE-pirA2B2并分别转入M15菌株表达,经SDS-PAGE和Western blot检测证明,3个重组菌株经IPTG诱导后,分别成功表达了可溶的PirA2,PirB2和PirA2B2蛋白。用亲和层析结合脱盐技术对3个重组菌株表达的外源蛋白分别进行纯化,并通过生物测定确定纯化蛋白的杀虫活性。【结果】生物测定结果显示联合表达的PirA2B2对大蜡螟和斜纹夜蛾五龄幼虫均有明显的血腔杀虫活性,LD50分别为每虫4.0和2.8μg,单独表达的PirA2或PirB2对上述2种害虫没有血腔杀虫活性,但两者的混合物具有与两者联合表达相似的杀虫活性;PirA2B2对大蜡螟和斜纹夜蛾初孵幼虫均无口服杀虫活性。【结论】pirA2B2是P.luminescens TT01菌株基因组中的另一个二元杀虫毒素基因。【意义】pirA2B2的成功克隆表达和杀虫功能的确定为进一步研究其与pirA1B1的关系以及该基因的表达调控等打下了基础。     

Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora and Its Bacterial Symbiont Photorhabdus luminescens

Entomoparasitic nematodes (EPNs) are being commercialized as a biocontrol measure for crop insect pests, as they provide advantages over common chemical insecticides. Mass production of these nematodes in liquid media has become a major challenge for commercialization. Producers are not willing to share the trade secrets of mass production and by doing so, have made culturing EPNs extremely difficult to advance existing technologies. Theoretically, mass production in liquid media is an ideal culturing method as it increases cost efficiency and nematode quantity. This paper will review current culturing methodologies and suggest basic culturing parameters for mass production. This review is focused on Heterorhabditis bacteriophora; however, this information can be useful for other nematode species.     

Improvement of Photorhabdus temperata bioinsecticides production in low‐cost media through adequate fermentation technology

To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H₂O₂ was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO‐shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 10⁸, 1.15 × 10⁸ cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

A Stilbene Optical Brightener can Enhance Bacterial Pathogenicity to Gypsy Moth (Lepidoptera: Lymantriidae) and Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Stilbene optical brighteners were first investigated to protect biological control agents such as viruses, fungi, and nematodes against ultraviolet light. Some are known to enhance the activity of insect viruses in Lepidoptera. In this work, one stilbene brightener, Tinopal LPW, also increased mortality of gypsy moth and Colorado potato beetle larvae when treated with bacteria/optical brightener combinations. This increase in mortality, however, did not occur for every bacteria/insect combination. In gypsy moth, a significant increase in larval mortality was observed only with Bacillus thuringiensis combined with Tinopal LPW. In Colorado potato beetle, however, the addition of Tinopal LPW increased larval mortality with all bacteria tested (B. thuringiensis, Serratia marcescens, Photorhabdus luminescens, and Chromobacterium sp.). The brightener also decreased the time to kill for these pathogens. This decrease in LT₅₀ was observed not only for bacteria+Tinopal LPW combinations, but also for combinations of Chromobacterium sp. toxin+Tinopal LPW. The mechanism for increase in bacterial toxicity by optical brighteners is compatible with mechanisms proposed for enhancement based on viral/lepidopteran/optical brightener systems that are not dependent on replication.     

Sequencing and Comparative Analysis of the lux Operon of Photorhabdus luminescens Strain Zm1: ERIC Elements as Putative Recombination Hot Spots

The EcoRI chromosomal fragment (6782 bp) containing the lux operon of Photorhabdus luminescens was cloned in pUC18 and completely sequenced. Enteric repetitive intergenic consensus (ERIC), an imperfect palindrome (125–127 bp) characteristic of Enterobacteriaceae genomes, was found in three sites. Strain Zm1 proved to differ in the ERIC number and location from strains Hb, Hm, and Hw. Nucleotide substitution analysis showed that luxC and luxB, which are more than 1 kb away from ERIC, are similar to the corresponding Hb genes, whereas luxD, luxA, and luxE, which are close to ERIC, are intermediate between their Hb and Hw counterparts. The Hb/Hw nucleotide substitution ratio was 1:1 in regions adjacent to ERIC. Hence ERIC can be thought to be recombination hot spots in the bacterial genome.     

Temperature Effects on Heterorhabditis megidis and Steinernema carpocapsae Infectivity to Galleria mellonella

The effect of temperature on the infection of larvae of the greater wax moth, Galleria mellonella, by Heterorhabditis megidis H90 and Steinernema carpocapsae strain All, was determined. For both species, infection, reproduction, and development were fastest at 20 to 24 °C. Infection by both H. megidis and S. carpocapsae occurred between 8 and 16 °C; however, neither species reproduced at 8 °C. Among the nematodes used in experiments at 8 °C, no H. megidis and very few S. carpocapsae developed beyond the infective juvenile stage. Compared with H. megidis, S. carpocapsae invaded and killed G. mellonella larvae faster at 8 to 16 °C. By comparing invasion rates, differences in infectivity between the two nematode species were detected that could not be detected in conventional petri dish bioassays where mortality was measured after a specified period. Invasion of G. mellonella larvae by H. megidis was faster at 24 than at 16 °C.     

Assessing the influence of the entomopathogenic nematode Heterorhabditis baujardi LPP7 (Rhabiditina) on embryogenesis and hatching of the plant-parasitic nematode Meloidogyne mayaguensis (Tylenchina)

Two assays were conducted to assess the influence of infective juveniles (IJs) of Heterorhabditis baujardi LPP7 on the embryogenesis and hatching of Meloidogyne mayaguensis. In the first assay, eggs were incubated in water alone or in the presence of infective juveniles, and completion of embryogenesis was evaluated 14 days later. In the second assay, unhatched second-stage juveniles were incubated in distilled water alone or in the presence of infective juveniles. Cumulative hatching was compared at various time intervals. Embryogenesis was not affected, whereas second-stage juveniles hatching was delayed probably because of the eggs permeability to noxious metabolites released by Photorhabdus luminescens, which is the bacterial symbiont of H. baujardi.