Frequent horizontal chromosome transfer between asexual fungal insect pathogens.

Entire chromosomes are typically only transmitted vertically from one generation to the next. The horizontal transfer of such chromosomes has long been considered improbable, yet gained recent support in several pathogenic fungi where it may affect the fitness or host specificity. To date, it is unknown how these transfers occur, how common they are, and whether they can occur between different species. In this study, we show multiple independent instances of horizontal transfers of the same accessory chromosome between two distinct strains of the asexual entomopathogenic fungus Metarhizium robertsii during experimental co-infection of its insect host, the Argentine ant. Notably, only the one chromosome-but no other-was transferred from the donor to the recipient strain. The recipient strain, now harboring the accessory chromosome, exhibited a competitive advantage under certain host conditions. By phylogenetic analysis, we further demonstrate that the same accessory chromosome was horizontally transferred in a natural environment between M. robertsii and another congeneric insect pathogen, Metarhizium guizhouense. Hence, horizontal chromosome transfer is not limited to the observed frequent events within species during experimental infections but also occurs naturally across species. The accessory chromosome that was transferred contains genes that may be involved in its preferential horizontal transfer or support its establishment. These genes encode putative histones and histone-modifying enzymes, as well as putative virulence factors. Our study reveals that both intra- and interspecies horizontal transfer of entire chromosomes is more frequent than previously assumed, likely representing a not uncommon mechanism for gene exchange.

Isolation and identification of bacteria from the invasive pest Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and evaluation of their biocontrol potential.

As an alternative to chemical insecticides, gut bacteria of insects could be used to control insect pests. In this study, bacteria associated with Tuta absoluta, an invasive species that has developed resistance to chemical insecticides, were isolated, and their potential for pest control was investigated. We isolated 13 bacteria from larvae of the pest and identified the isolates on the basis of their morphological, physiological, biochemical, and molecular characteristics as Bacillus thuringiensis (Ta1-8), Staphylococcus petrasii (Ta9), Citrobacter freundii (Ta10), Chishuiella changwenlii (Ta11), Enterococcus casseliflavus (Ta12), and Pseudomonas tremae (Ta13). A laboratory screening test at 109 cfu/ml showed that B. thuringiensis (Bt) isolates caused more than 90% mortality after 3 days. Among the isolates, Bt-Ta1 showed the highest mortality in a short time. The LC50 and LC90 values for Bt-Ta1 were estimated to be 1.2 × 106 and 2 × 109 cfu/ml, respectively. Detailed characterization of Bt-Ta1 revealed that it is one of the serotypes effective on lepidopterans and contains the genes cry1Aa, cry2Aa, and vip3Aa, which encode lepidopteran toxic proteins. Bt-Ta1 isolate has been shown to have the potential to be used in the integrated management of Tuta absoluta.

Influences of compound age and identity in the effectiveness of insect quinone secretions against the fungus Beauveria bassiana.

Chemical defences against parasites and pathogens can be seen in a wide range of animal taxa, including insect pests such as the red flour beetle Tribolium castaneum. Antimicrobial quinone-based secretions can be used by these beetles to defend against various parasites, particularly the fungal entomopathogen Beauveria bassiana. While quinone secretions can inhibit B. bassiana growth, it is unknown how long they remain effective or how individual secretion compounds contribute to growth inhibition. Here, we tested each individual component of the quinone secretions (methyl-1,4-benzoquinone, ethyl-1,4-benzoquinone, and 1-pentadecene), as well as two mixed solutions that represent the composition range found in natural T. castaneum secretions, after aging for 0, 24, or 72 h. The two quinone compounds equally contributed to B. bassiana inhibition, but their efficacy was significantly reduced after 24 h, with no growth inhibition after 72 h. This indicates that quinones protect insects against B. bassiana for only a limited time, perhaps requiring constant secretion into the environment to effectively defend against this fungal threat. Future investigations may consider the extent to which quinone secretions are effective against other parasites, as well as how their ability to cause parasite damage changes with compound age.

Evaluation of the effectiveness of two Iranian su-strains of Metarhizium anisopliae (ascomycota: hypocreales) on the mortality rate of American cockroach.

The cockroach is one of the most important disease vectors in world. Entomopathogenic fungi, as three concentrations of spores were taken 1.1 × 105, 1.1 × 107, and 1.1 × 109 conidia/mL from two isolates of Nour and Saravan-Iranian. In this study, the immersion method caused about 13% mortality only in isolation (1 × 109 conidia/mL) of Saravan isolates. Inoculation of isolates below the pronotum did not significantly differ the mortality rate between the two genera (P = 0.8), compared to the pathogenicity of three isolates of M. anisopliae (1.1 × 105, 1.1 × 107, and 1.1 × 109 conidia/mL). In total, Saravan and Nour isolates were 66%, 73%, and 93%, respectively, indicating a significant difference (P < 0.001). Mortality of male and female cockroaches with Saravan isolates respectively occurred 3 and 4 days after inoculation (LT50 = 4.3d), while for Nour isolates, in both sexes, mortality was observed within four days after the test (LT50 = 5.5d). Considering the results M. anisopliae can be one benefit methods for control American cockroach in the future. .

Genetic polymorphism and plant growth promotion traits of potent fungal entomopathogens of rice leaf folder.

Entomopathogenic fungal biocides are preferred for environment friendly sustainable management of insect pests due to their host specificity and harmlessness to non-target insects. Plant growth promotion (PGP) functions of the entomofungi are also important attributes but hitherto insignificantly explored. Therefore, virulence of 17 natural fungal entomocides (Cordyceps, Beauveria, Metarhizium, Nomuraea, Fusarium, Verticillium, Trichoderma and Paecilomyces spp.) were evaluated for pathogenicity against five rice pests (brown plant hopper (Nilaparvata lugens) and green leaf hopper (Nephotettix virescens) nymphs, leaf folder (Cnaphalocrosis medinalis) and yellow stem borer (Scirpophaga incertulas) larvae and swarming caterpillar (Spodoptera mauritia), respectively), and PGP traits of the potent leaf folder pathogens. Among the fungi, only the leaf folder pathogens (3 isolates each of Beauveria and Metarhizium spp.) infected > 50% (80-90%) larvae but other fungi were ineffective as infected < 50% (0-47%) insects. Besides, the leaf folder pathogens exhibited diverse PGP traits such as organic/inorganic phosphate solubilization (104.7-236.4 µg/ml), and siderophore, ammonia, hydrogen cyanide (HCN), indole production etc. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequences repeat (SSR) and internal transcribed spacers (ITS) analysis ascertained strain identity and genetic (inter and intra-specific) diversity among the potent biocides Beauveria and Metarhizium spp. The virulent natural fungal pathogens of rice pests with polyvalent PGP traits may be prospected for rice growth promotion and biocontrol of leaf folder.

Subterranean termites raise the alarm when their anti-fungal weapon falters.

Termicin is an anti-fungal defensin that is disseminated from termite salivary glands. The peptide appears to be critical for the elimination with mutual grooming (allogrooming) of pathogenic spores (conidia) that have attached to the insect cuticle. There has been a recent selective sweep for an advantageous variant of this peptide in the subterranean termite Reticulitermes flavipes. We tested the anti-mycotic activity of a recombinant termicin corresponding with this variant against the conidia of different Metarhizium fungal isolates from soil close to foraging R. flavipes workers. Termicin was most effective against isolates that had previously been shown to elicit a relatively weak alarm response, as indicated by brief bouts of rapid longitudinal oscillatory movement (LOM). These isolates that elicited weak alarm were also the deadliest apparently because the survival of termites exposed to the fungus depends on a strong social immune response (LOMs and allogrooming). The selective pressure for a single termicin variant may have been driven by the most dangerous isolates that elicit a weak behavioral response. The correlation between termicin anti-fungal activity and LOM suggests that pathogen-associated molecular patterns that affect termite recognition of conidial contamination and the onset of elevated allogrooming also affect the vulnerability of conidia to the disruption of their cell membranes by termicin.

Microsclerotial pellets of Metarhizium spp.: thermotolerance and bioefficacy against the cattle tick.

The cattle tick, Rhipicephalus microplus (Acari: Ixodidae), is a multi-billion dollar ectoparasite of global importance affecting beef and milk production. Submerged cultures of cosmopolitan entomopathogenic fungal species of the genus Metarhizium typically produce microsclerotia that provide both long-term survival and environmental resistance. Microsclerotia hold great potential as an unconventional active propagule to control this tick under laboratory and semi-field conditions. However, heat stress caused especially by elevated temperatures poses a critical environmental constraint for the successful development and efficacy of microsclerotia under tropical conditions. First, we screened six strains of Metarhizium anisopliae, Metarhizium robertsii and Metarhizium humberi for their ability to produce microsclerotia by submerged liquid cultivation. In addition, we assessed the biological fitness and bioefficacy of dried microsclerotial pellets under amenable (27 °C) and heat-stressed (32 °C) incubation against engorged adult females of R. microplus. Microsclerotia in pelletized formulation prepared with carriers based on diatomaceous earth and microcrystalline cellulose exhibited conidial production at different extents according to the fungal strain and the incubation temperature, but most strains displayed reduced sporogenesis when exposed to 32 °C. Engorged tick females exposed to sporulated microsclerotia from pelletized M. anisopliae CG47 or IP 119 had fewer number of hatching larvae in comparison to the control group, irrespective of the incubation temperature tested. The minimum dosage of microsclerotial pellets that effectively reduced hatchability of tick larvae was estimated to be 2 mg per plate (equivalent to 6.0 kg per hectare). Metarhizium microsclerotial pellets exhibited significant tolerance to 32 °C and pronounced acaricidal activity against this economically important ectoparasite of cattle, even under simulated environmental heat stress. KEY POINTS: • Heat stress affects conidial production by microsclerotia of most pelletized Metarhizium strains • Heat stress does not impair the acaricidal performance of pelletized microsclerotia • Pellet formulation of Metarhizium microsclerotia is a promising mycoacaricide.

Synergistic interactions between three insecticides and Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) in lesser mealworm, Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae), larvae.

Topical applications of insecticides ß-cyfluthrin, imidacloprid, and spinosad in combination with Beauveria bassiana (topical and contact filter paper application) induced synergistic interactions in lesser mealworm larvae, increasing mortality and in some cases numbers of larval cadavers exhibiting conidiogenesis. Reduced concentrations (LC10, LC20, LC30) of the insecticides induced sublethal effects in lesser mealworm larvae, inhibiting development (mass, head-capsule width, moulting) after eight days' exposure and movement behaviour (area explored and distance travelled) after 3 h' exposure. The most potent synergist was ß-cyfluthrin, it strongly inhibited larval development and movement while significantly increasing mortality and conidiogenesis in B. bassiana-infected larvae. Imidacloprid also strongly inhibited larval development and movement, but only produced weak short-lived synergistic increases in mortality, with no increase in conidiogenesis. Spinosad induced no effect on development and limited effect on movement, but still induced moderate short-lived synergistic increases in mortality and conidiogenesis. Intoxicated larvae exposed to B. bassiana on filter paper for 3 h showed no synergistic interactions, except when intoxicated by spinosad.

Beauveria australis finds a new host in French dung beetles introduced to Australia.

Exotic dung beetles have been introduced to Australia for over 50 years to mitigate issues caused by dung produced by livestock. This study aims at identifying fungi affecting a beetle colony and investigating their source. Fungal hyphae emerging from the cuticle of dead beetles were cultured and a multigene phylogeny showed that Beauveria bassiana and B. australis both infected these introduced beetles, likely acquired through local soil or dung. This is the first record of B. australis infecting family Scarabaeidae. This study highlights the importance of fungal outbreak management in insect rearing before release, and challenges associated with new pathogens.

Enhanced toxicity of entomopathogenic fungi Beauveria bassiana with bacteria expressing immune suppressive dsRNA in a leaf beetle.

The entomopathogenic fungus is recognized as an ideal alternative to chemical pesticides, nonetheless, its efficacy is often limited by insect's innate immune system. The suppression of the host immunity may overcome the obstacle and promote the toxicity of the fungi. Here, by using an entomopathogenic fungus Beauveria bassiana and immune genes dsRNA-expressing bacteria, we explored the potentially synergistic toxicity of the two agents on a leaf beetle Plagiodera versicolora (Coleoptera: Chrysomelidae). We first determined the susceptibilities of P. versicolora to a B. bassiana 476 strain (hereafter referred to Bb476). And the immune genes were identified based on the transcriptome of Bb476 challenged beetles. Subsequently, five immune genes (PGRP1, Toll1, Domeless，SPN1，and Lysozyme) were targeted by feeding dsRNA-expressing bacteria, which produced a 71.4, 39.0, 72.0, 49.0, and 68.7% gene silencing effect, respectively. Furthermore, we found a significantly increased mortality of P. versicolora when combined the Bb476 and the immune suppressive dsRNAs. Taking together, this study highlights the importance of insect immunity in the defense of entomopathogens and also paves the way toward the development of a more efficient pest management strategy that integrates both entomopathogens and immune suppressive dsRNAs.

First record of Fusarium concentricum (Hypocreales: Hypocreaceae) isolated from the moth Polychrosis cunninhamiacola (Lepidoptera: Tortricidae) as an entomopathogenic fungus.

Fusarium concentricum Nirenberg & O' Donnell (Ascomycota: Hypocreales) is a fungal species known to infect plants, but never reported as entomopathogenic. Polychrosis cunninhamiacola Liu et Pei (Lepidoptera: Tortricidae: Olethreutinae) is a major and widespread insect pest causing economic losses to cultivated Chinese fir Cunninghamia lanceolata (Lamb.) Hook. It is routinely controlled by extensive use of chemical insecticides, which is perceived as environmentally unsustainable. During March and April of 2019-2020, muscardine cadavers of larvae and pupae of P. cunninhamiacola infected with growing fungus were collected in a fir forest in northern Guangdong Province, China. Conidia were isolated and cultured on PDA medium, from which the fungal strain was identified as F. concentricum FCPC-L01 by morphology and by sequence alignment match with Tef-1α gene. Pathogenicity bioassays at the conidial concentration 1 × 107 revealed P. cunninhamiacola adults and Danaus chrysippus (L.) (Lepidoptera: Nymphalidae) larvae are sensitive to the fungal infection, but not the fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae). We believe results indicate this fungal strain might be applicable against specific target insect pests. As this is the first record of a natural infection caused by F. concentricum in insects, we propose host specificity tests should be done to evaluate its potential as a biocontrol agent.

Multiple S-Layer Proteins of Brevibacillus laterosporus as Virulence Factors against Insects.

S-layers are involved in the adaptation of bacteria to the outside environment and in pathogenesis, often representing special virulence factors. Vegetative cells of the entomopathogenic bacterium Brevibacillus laterosporus are characterized by an overproduction of extracellular surface layers that are released in the medium during growth. The purpose of this study was to characterize cell wall proteins of this bacterium and to investigate their involvement in pathogenesis. Electron microscopy observations documented the presence of multiple S-layers, including an outermost (OW) and a middle (MW) layer, in addition to the peptidoglycan layer covering the plasma membrane. After identifying these proteins (OWP and MWP) by mass spectrometry analyses, and determining their gene sequences, the cell wall multilayer-released fraction was successfully isolated and used in insect bioassays alone and in combination with bacterial spores. This study confirmed a central role of spores in bacterial pathogenicity to insects but also detected a significant virulence associated with fractions containing released cell wall multilayer proteins. Taken together, S-layer proteins appear to be part of the toxins and virulence factors complex of this microbial control agent of invertebrate pests.

Genome sequence of the entomopathogenic Serratia entomophila isolate 626 and characterisation of the species specific itaconate degradation pathway.

BACKGROUND: Isolates of Serratia entomophila and S. proteamaculans (Yersiniaceae) cause disease specific to the endemic New Zealand pasture pest, Costelytra giveni (Coleoptera: Scarabaeidae). Previous genomic profiling has shown that S. entomophila isolates appear to have conserved genomes and, where present, conserved plasmids. In the absence of C. giveni larvae, S. entomophila prevalence reduces in the soil over time, suggesting that S. entomophila has formed a host-specific relationship with C. giveni. To help define potential genetic mechanisms driving retention of the chronic disease of S. entomophila, the genome of the isolate 626 was sequenced, enabling the identification of unique chromosomal properties, and defining the gain/loss of accessory virulence factors relevant to pathogenicity to C. giveni larvae. RESULTS: We report the complete sequence of S. entomophila isolate 626, a causal agent of amber disease in C. giveni larvae. The genome of S. entomophila 626 is 5,046,461 bp, with 59.1% G + C content and encoding 4,695 predicted CDS. Comparative analysis with five previously sequenced Serratia species, S. proteamaculans 336X, S. marcescens Db11, S. nematodiphila DH-S01, S. grimesii BXF1, and S. ficaria NBRC 102596, revealed a core of 1,165 genes shared. Further comparisons between S. entomophila 626 and S. proteamaculans 336X revealed fewer predicted phage-like regions and genomic islands in 626, suggesting less horizontally acquired genetic material. Genomic analyses revealed the presence of a four-gene itaconate operon, sharing a similar gene order as the Yersinia pestis ripABC complex. Assessment of a constructed 626::RipC mutant revealed that the operon confer a possible metabolic advantage to S. entomophila in the initial stages of C. giveni infection. CONCLUSIONS: Evidence is presented where, relative to S. proteamaculans 336X, S. entomophila 626 encodes fewer genomic islands and phages, alluding to limited horizontal gene transfer in S. entomophila. Bioassay assessments of a S. entomophila-mutant with a targeted mutation of the itaconate degradation region unique to this species, found the mutant to have a reduced capacity to replicate post challenge of the C. giveni larval host, implicating the itaconate operon in establishment within the host.

Alkane-priming of Beauveria bassiana strains to improve biocontrol of the redbanded stink bug Piezodorus guildinii and the bronze bug Thaumastocoris peregrinus.

Insect epicuticle hydrocarbons (CHC) are known to be important determinants in the susceptibility degree of insects to fungal entomopathogens. Five Beauveria bassiana (Balsamo) Vuillemin (Hypocreales; Clavicipitaceae) strains were phenotypically analyzed regarding their response to CHC nutrition and their pathogenicity and virulence towards high fungal-susceptible Thaumastocoris peregrinus (Carpintero and Dellapé) (Heteroptera: Thaumastocoridae) and low fungal-susceptible Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae), which are important hemipteran pests in eucalyptus and soybean plantations, respectively. Two of these strains, which were the most (ILB308) and the least (ILB299) virulent to P. guildinii, were also evaluated at gene expression level after growth on n-pentadecane, a P. guildinii epicuticular hydrocarbon. Beauveria bassiana hypervirulent strain ILB308 showed the lowest growth on most evaluated CHC media. However, this strain distinctively induced most of the analyzed genes involved in CHC assimilation, cuticle degradation and stress tolerance. Virulence towards low susceptibility P. guildinii was enhanced in both hypervirulent ILB308 and hypovirulent ILB299 strains after growth on n-pentadecane as the sole carbon source, whereas virulence enhancement towards high susceptibility T. peregrinus was only observed in the hypervirulent strain. Virulence enhancement towards P. guildinii could be mostly explained by a priming effect produced by CHC on the induction of some genes related to hydrocarbon assimilation in ILB299 and ILB308, such as cytochrome P450 genes (BbCyp52g11 and BbCyp52x1), together with adhesion and stress tolerance genes, such as hydrophobin (Bbhyd2) and catalase (Bbcatc) and glutathione peroxidase (Bbgpx), respectively.

Lack of avoidance of the fungal entomopathogen, Metarhizium brunneum, by male Agriotes obscurus beetles.

Fungal entomopathogens can greatly reduce the fitness of their hosts, and it is therefore expected that susceptible insects will be selected to avoid exposure to pathogens. Metarhizium brunneum is a fungal pathogen that can infect Agriotes obscurus, which in its larval form is a destructive agricultural pest and is repelled by the presence of M. brunneum conidia. Due to the subterranean nature of larval A. obscurus, recent research has focused on targeting adult A. obscurus with M. brunneum. No-choice and choice behavioural assays were conducted to determine if male adult A. obscurus avoid M. brunneum mycosed cadavers, or conidia applied to either food or soil. To further investigate the response of A. obscurus beetles to conspecific cadavers, the movement and behaviour of beetles placed at the centre of a semi-circular arrangement of mycosed or control cadavers was examined using motion tracking software. We found little evidence to suggest that A. obscurus male beetles avoid M. brunneum conidia or mycosed conspecific cadavers or alter their behaviour in their presence.

An eco-friendly approach of biocontrol of aphid (Aphis gossypii Glover) by Trichoderma harzianum.

Aphid (Aphis gossypii) is one of the important pests of papaya crop. In this work, applications of Trichoderma harzianum and Beauveria bassiana (biocontrol agents) and malathion (insecticide) were conducted in vitro and in agrifields for testing their anti-aphid efficacy and compared their efficacy. Furthermore, the enzymatic mechanism of T. harzianum with respect to biocontrolling the pest was unearthed. The LD50 dose of T. harzianum and B. bassiana was 1.2 × 105 spores mL-1 and 1.0 × 106 spores mL-1 respectively after 48 h of administration. The LT50 of T. harzianum also exhibited a lower effective time (47.70 h) than B. bassiana (57.53 h) for the same concentration of spores applied (1 × 105 spores mL-1). The pooled data analysis of two years (2019-2020) showed that the application of T. harzianum spores in agrifields exhibited 31.75 ± 13.00a percentage of reduction of aphid population whereas malathion exhibited 23.93 ± 1.30a%, in comparison to control. The statistical analysis indicated that the application of malathion exhibited the same efficacy as T. harzianum isolate and placed in the same category. In plate detection assay, T. harzianum produced a higher hydrolytic zone for chitinase (8.0 ± 0.4 cm diameter) and protease (7.0 ± 0.4 cm diameter) enzymes, than B. bassiana (1.3 ± 0.2 cm and 1.1 ± 0.2 cm respectively). Quantitative estimation of enzymes exhibited that T. harzianum produced 299 ± 11a µg mL-1 of chitinase, 519 ± 19a µg mL-1 of protease, and 65 ± 12a µg mL-1 of PR1, and on the other hand, B. bassiana yielded 124 ± 12b, 361 ± 23b, and 29 ± 18b µg mL-1 of chitinase, protease, and PR1 respectively. It indicated that T. harzianum was superior over the B. bassiana in terms of production capacity of all three enzymes. In conclusion, all the above experimental results suggested that T. harzianum showed better aphid-killing efficacy than B. bassiana. It also suggested that T. harzianum should replace hazardous chemical pesticide (malathion) for eco-friendly biocontrol of aphids.

Immunological interactions of Chilo suppressalis Walker (Lepidoptera: Crambidae) with the native entomopathogenic fungi.

Entomopathogenic fungi can attack many insect hosts and have been applied as the eco-friendly alternatives to synthetic chemicals for the control of pests. Insects have developed different defense systems encountering entomopathogens including humoral and cellular immune responses. In the present study, injection of some native entomopathogenic fungi to the Chilo suppressalis Walker larvae resulted in an enhancement of the cellular and antimicrobial defenses. The numbers of total and differential hemocytes increased rapidly in the first 3 and 6 h but those gradually reduced 12 and 24 h post-injections. The nodule formation and phenoloxidase activity increased at the time intervals after fungal infection. A similar trend was found in the transcription of antimicrobial peptides including attacin1 and 2, cecropin1 and 2, gallerimycin, defensin, lysozyme, and prophenoloxidase-activating proteinase-3 during infection fungi. In all cases, the target gene transcription was upper in the larvae injected by the fungi than that of control larvae. These results may elucidate better knowledge on the interaction of the fungi present in agroecosystems with the target insect pest.

Identification and phylogenetic analysis of a collection of Beauveria spp. Isolates from Central America and Puerto Rico.

The genus Beauveria comprises economically important entomopathogenic fungi, widely used for biological control in agriculture. Interest in these organisms in Costa Rica prompted surveys and establishment of collections in the past two decades. However, there was neither a formal identification nor a characterization of the isolates. With that purpose, the morphology and genetic variation by microsatellites and partial sequencing of Bloc, TEF-1α and RPB2 regions were studied for 32 isolates of Beauveria, which included 26 from Costa Rica, five from Puerto Rico and one from Honduras. The isolates were identified as B. bassiana (29) and B. caledonica (3). Ninety-three percent of B. bassiana isolates belonged to a monophyletic group of African and Neotropical isolates. A total of 105 alleles were recorded with 11 SSR markers, and the results suggested high diversity within the collection. Mantel tests showed low association between geographic origin and the variation among isolates.

Bacillus pumilus 15.1, a Strain Active against Ceratitis capitata, Contains a Novel Phage and a Phage-Related Particle with Bacteriocin Activity.

A 98.1 Kb genomic region from B. pumilus 15.1, a strain isolated as an entomopathogen toward C. capitata, the Mediterranean fruit fly, has been characterised in search of potential virulence factors. The 98.1 Kb region shows a high number of phage-related protein-coding ORFs. Two regions with different phylogenetic origins, one with 28.7 Kb in size, highly conserved in Bacillus strains, and one with 60.2 Kb in size, scarcely found in Bacillus genomes are differentiated. The content of each region is thoroughly characterised using comparative studies. This study demonstrates that these two regions are responsible for the production, after mitomycin induction, of a phage-like particle that packages DNA from the host bacterium and a novel phage for B. pumilus, respectively. Both the phage-like particles and the novel phage are observed and characterised by TEM, and some of their structural proteins are identified by protein fingerprinting. In addition, it is found that the phage-like particle shows bacteriocin activity toward other B. pumilus strains. The effect of the phage-like particles and the phage in the toxicity of the strain toward C. capitata is also evaluated.

The Combination of Bacillus Thuringiensis and Its Engineered Strain Expressing dsRNA Increases the Toxicity against Plutella Xylostella.

RNA interference (RNAi) has been developed and used as an emerging strategy for pest management. Here, an entomopathogen Bacillus thuringiensis (Bt) was used to express the dsRNA for the control of Plutella xylostella. A vector containing a 325-bp fragment of the conserved region of P. xylostella arginine kinase gene (PxAK) flanking in two ends with the promoter Pro3α was developed and transferred into Bt 8010 and BMB171, and consequently engineered Bt strains 8010AKi and BMB171AKi expressing dsRNA of PxAK were developed. The two engineered Bt strains were separately mixed with Bt 8010 in a series of ratios, and then fed to the P. xylostella larvae. We found that 8010:8010AKi of 9:1 and 8010:BMB171AKi of 7:3 caused a higher mortality than Bt 8010. PxAK expression levels in the individuals treated with the mixtures, 8010AKi and BMB171Aki, were lower than that in the control. The intrinsic rate of increase (r) and net reproductive rate (R0) of the population treated with 8010:8010AKi of 9:1 were lower than those of the population treated with Bt 8010 or 8010AKi. We developed a Bt-mediated insect RNAi for the control of P. xylostella and demonstrated a practical approach to integrating the entomopathogen with RNAi technique for the pest management.