Laboratory evaluation of the acute and chronic toxicity of diflubenzuron against IV instar Mediterranean locust Dociostaurus maroccanus (Thunberg)

The lethal effects of the benzoylphenyl urea diflubenzuron were assessed under laboratory conditions against one day old fourth (IV) Dociostaurus maroccanus (Thunberg) (Orthoptera: Acrididae: Gomphocerinae) instars. Following exposure by ingestion of a single dose applied to short pieces of wheat seedlings, nymphs were monitored for two moults until the adult stage. The obtained acute response gave an LD50 of 1.47 μg a.i./nymph. The male and female nymphs response to diflubenzuron did not show statistical significance but there was a narrow tolerance range within the female treated population. Analysis of the relative mortality in classes at individual dose rates showed that mortality occurring in the moult to V instar increased directly related to diflubenzuron dose rate. The duration of IV instar was significantly higher following treatments with all diflubenzuron doses, whereas the duration of the V instar did not show any clear differences between treated and control nymphs. The main sublethal effects on these adult females was a reduction of the number eggs per egg-pods, which could have major impacts on the evolution of D. maroccanus outbreaks and their management. Key words: Moroccan locust, locust control, lethal effects, sublethal effects, reproduction. VALUTAZIONI DI LABORATORIO SULLA TOSSICITÀ ACUTA E CRONICA DEL DIFLUBENZURON CONTRO NINFE DELLA CAVALLETTA MEDITERRANEA DociostAurus MAroccAnus (THUNBERG) Sono stati valutati mediante saggi di laboratorio gli effetti letali di benzoylphenyl urea diflubenzuron contro ninfe di Dociostaurus maroccanus (Thunberg) (Orthoptera: Acrididae: Gomphocerinae). Dosi singole sono state applicate su pezzetti di cariossidi di grano e, dopo l’ingestione, la vita delle ninfe è stata monitorata fino al raggiungimento dello stadio adulto. I dati ottenuti hanno evidenziato una DL50 di 1.47 μg a.i./ninfa. La risposta delle ninfe maschio e femmina al diflubenzuron non ha evidenziato alcun significato statistico anche se la popolazione delle femmine trattate ha mostrato una certa tolleranza. L’analisi della mortalità relativa ad ogni singola dose utilizzata ha evidenziato che la mortalità ottenuta nel passaggio al secondo stadio di ninfa aumentava in maniera direttamente proporzionale alla dose utilizzata. La durata del I stadio ninfale è stata significativamente maggiore dopo il trattamento con diflubenzuron a tutte le dosi, mentre la durate del II stadio ninfale non ha mostrato alcuna differenza tra ninfe trattate e non trattate. Il maggiore effetto subletale sulle femmine adulte è stato la riduzione del numero di uova che ha influenzato il successivo sviluppo della popolazione di D. maroccanus. Parole chiave: Dociostauro, controllo, effetti letali, effetti subletali, riproduzione

Delving into the Causes and Effects of Entomopathogenic Endophytic Metarhizium brunneum Foliar Application-Related Mortality in Spodoptera littoralis Larvae

The aim of the current study was to delve into the causes of mortality of Spodoptera littoralis larvae feeding on Metarhizium-colonized plants in the absence of fungal outgrowth on the cadavers as previous studies reported and to elucidate the possible indirect effects of this fungus-colonized diet. The effect was evaluated in experiments conducted using leaf discs of colonized plants and in planta using fungus-colonized whole plants. The mortality rates of larvae fed on Metarhiziumcolonized melon leaves were 45.0% and 87.5%, and the average survival times were 6.6 and 3.1 days in experiments performed with discs and in planta, respectively. Notably, these mortality levels were not associated with observed apoptosis mediated by caspases 1, 3-7 and 8; thus, further investigation into the possible immune system reaction of the insect after the ingestion of colonized plants is required. The leaf consumption of S. littoralis larvae fed on melon-colonized leaves was lower than that on control plants in the disc experiments but not in experiments conducted in planta. In this regard, in experiments performed in planta, plant damage increased larval mortality in both fungally challenged and control larvae. There was also a meaningful effect of exposure to Metarhiziumcolonized melon leaf discs on S. littoralis fitness, with significant reductions in 39.0% and 22.0% in female fecundity and egg fertility, respectively, detected in females emerging from pupae developing from larvae surviving exposure to colonized plant discs; all larvae died in the in planta experiments. Hence, the present work presents new findings revealing the high potential of endophytic entomopathogenic fungi to improve the outcome of foliar applications against chewing insects in the short, mid- and long term, by the reduction of the reproductive potential of surviving adults and reveals new insights into the development of bioassays with whole plants for more detailed evaluation of the impact of these fungi as endophytes used for plant protection

Production of Microsclerotia by Metarhizium sp., and Factors Affecting Their Survival, Germination, and Conidial Yield

Microsclerotia (MS) produced by some species of Metarhizium can be used as active ingredients in mycoinsecticides for the control of soil-dwelling stages of geophilic pests. In this study, the MS production potential of two Metarhizium brunneum strains and one M. robertsii strain was evaluated. The three strains were able to produce MS in liquid fermentation, yielding between 4.0 × 106 (M. robertsii EAMa 01/158-Su strain) and 1.0 × 107 (M. brunneum EAMa 01/58-Su strain) infective propagules (CFU) per gram of MS. The EAMa 01/58-Su strain was selected for further investigation into the effects of key abiotic factors on their survival and conidial yield. The MS were demonstrated to be stable at different storage temperatures (−80, −18, and 4 °C), with a shelf-life up to one year. The best temperature for MS storage was −80 °C, ensuring good viability of MS for up to one year (4.9 × 1010 CFU/g MS). Moreover, soil texture significantly affected CFU production by MS; sandy soils were the best driver of infective propagule production. Finally, the best combination of soil temperature and humidity for MS germination was 22.7 °C and 7.3% (wt./wt.), with no significant effect of UV-B exposure time on MS viability. These results provide key insights into the handling and storage of MS, and for decision making on MS dosage and timing of application

Effects of Endophytic Entomopathogenic Ascomycetes on the Life-History Traits of Aphis gossypii Glover and Its Interactions with Melon Plants

Entomopathogenic fungi are sprayed commercially for aphid control in greenhouses. Recently, their ability to grow endophytically within plants was discovered, offering the opportunity for systemic biological control. Endophytic colonization of host plants could also influence life-table parameters and behavior of herbivores. We investigated lethal and pre-mortality effects of Beauveria bassiana and Metarhizium brunneum on Aphis gossypii; aphids either received inoculum while feeding on recently sprayed leaves (surface inoculum and endophytically-colonized) or while feeding on unsprayed but endophytically-colonized leaves. We used choice assays to identify any preferences for endophytically-colonized or control plants. Volatile emissions from endophytically-colonized plants and control plants were also compared. Aphid mortality rates ranged between 48.2 and 56.9% on sprayed leaves, and between 37.7 and 50.0 on endophytically-colonized leaves. There was a significant effect of endophytic colonization on the rate of nymph production, but this did not result in an overall increase in the aphid population. Endophytic colonization did not influence host-plant selection even though there were qualitative and quantitative differences in the blend of volatiles released by endophytically-colonized and control plants. Although endophytic colonization did not change herbivore behavior, plants still benefit via indirect defense, resistance to plant pathogens or abiotic stress tolerance

Effect of passage through the plant on virulence and endophytic behavioural adaptation in the entomopathogenic fungus Beauveria bassiana

Systemic crop protection using endophytic isolates of entomopathogenic fungi is at the forefront of IPM. Nonetheless, any potential trade-offs between virulence and endophytic behaviour must be elucidated if they are to be effectively used in pest management strategies. Here we investigated endophytic adaptation in an isolate of Beauveria bassiana following successive passage through melon, tomato and cotton tissues. Plants were sprayed with a suspension of B. bassiana endophytic isolate EABb 04/01-Tip to initiate endophytic colonization. Once colonization was established, the fungus was re-isolated from the plant, applied to another plant and re-isolated again; this was repeated to achieve three passages. After each passage, a conidial suspension of each isolate was used in bioassays to evaluate both virulence against 4th instar larvae of the model insect Galleria mellonella and to quantify the extent of endophytic activity in each respective host plant species. When sprayed leaves were inspected for fungal colonization, differences in percentage tissue colonization amongst the plant species were detected after the first re-isolation. Endophytic colonization rates in melon and tomato, which varied from 70 to 100%, were higher than those observed in cotton, which ranged from 40 to 50%; endophytic colonization in cotton increased to 75–100% after the third passage. This improvement in endophytic behaviour in cotton, an apparently suboptimal plant for fungal colonization, suggests an evolutionary adaptation to localized or transient endophytic colonization, while further assays are needed. Meanwhile, when endophytic colonization of non-sprayed leaves distant from the sprayed ones was investigated, endophytic activity was evident in all three crop species suggesting that movement within plants after successive passage increased the extent of endophytic colonization from transient to systemic. The present research highlights the potential for adaptation to endophytic behaviour in crops that are less suitable for endophytic colonization. Furthermore, we demonstrated stability in virulence after multiple passages through host plants. This is a key result for the development of IPM strategies based on endophytic entomopathogenic fungi

Entomopathogenic Fungi-Mediated Solubilization and Induction of Fe Related Genes in Melon and Cucumber Plants

Endophytic insect pathogenic fungi have a multifunctional lifestyle; in addition to its well-known function as biocontrol agents, it may also help plants respond to other biotic and abiotic stresses, such as iron (Fe) deficiency. This study explores M. brunneum EAMa 01/58-Su strain attributes for Fe acquisition. Firstly, direct attributes include siderophore exudation (in vitro assay) and Fe content in shoots and in the substrate (in vivo assay) were evaluated for three strains of Beauveria bassiana and Metarhizium bruneum. The M. brunneum EAMa 01/58-Su strain showed a great ability to exudate iron siderophores (58.4% surface siderophores exudation) and provided higher Fe content in both dry matter and substrate compared to the control and was therefore selected for further research to unravel the possible induction of Fe deficiency responses, Ferric Reductase Activity (FRA), and relative expression of Fe acquisition genes by qRT-PCR in melon and cucumber plants.. In addition, root priming by M. brunneum EAMa 01/58-Su strain elicited Fe deficiency responses at transcriptional level. Our results show an early up-regulation (24, 48 or 72 h post inoculation) of the Fe acquisition genes FRO1, FRO2, IRT1, HA1, and FIT as well as the FRA. These results highlight the mechanisms involved in the Fe acquisition as mediated by IPF M. brunneum EAMa 01/58-Su strain

Genetic analyses place most Spanish isolates of Beauveria bassiana in a molecular group with word-wide distribution

<p>Abstract</p> <p>Background</p> <p>The entomopathogenic anamorphic fungus <it>Beauveria bassiana </it>is currently used as a biocontrol agent (BCA) of insects. Fifty-seven <it>Beauveria bassiana </it>isolates -53 from Spain- were characterized, integrating group I intron insertion patterns at the 3'-end of the nuclear large subunit ribosomal gene (LSU rDNA) and elongation factor 1-alpha (EF1-α) phylogenetic information, in order to assess the genetic structure and diversity of this Spanish collection of <it>B. bassiana</it>.</p> <p>Results</p> <p>Group I intron genotype analysis was based on the four highly conserved insertion sites of the LSU (Ec2653, Ec2449, Ec2066, Ec1921). Of the 16 possible combinations/genotypes, only four were detected, two of which were predominant, containing 44 and 9 members out of 57 isolates, respectively. Interestingly, the members of the latter two genotypes showed unique differences in their growth temperatures. In follow, EF1-α phylogeny served to classify most of the strains in the <it>B. bassiana s.s</it>. (<it>sensu stricto</it>) group and separate them into 5 molecular subgroups, all of which contained a group I intron belonging to the IC1 subtype at the Ec1921 position. A number of parameters such as thermal growth or origin (host, geographic location and climatic conditions) were also examined but in general no association could be found.</p> <p>Conclusion</p> <p>Most Spanish <it>B. bassiana </it>isolates (77.2%) are grouped into a major phylogenetic subgroup with word-wide distribution. However, high phylogenetic diversity was also detected among Spanish isolates from close geographic zones with low climatic variation. In general, no correlation was observed between the molecular distribution and geographic origin or climatic characteristics where the Spanish <it>B. bassiana </it>isolates were sampled.</p

Survey of Natural Enemies of the Invasive Boxwood Moth Cydalima perspectalis in Southwestern Mediterranean Europe and Biocontrol Potential of a Native Beauveria bassiana (Bals.-Criv.) Vuill. Strain

Cydalima perspectalis (Lepidoptera: Crambidae), a species native to East Asia, has been especially devastating in the Mediterranean region and Catalonia, northeast Spain, where Buxus sempervirens is an essential component of the natural forest. As an invasive species, the lack of biotic mortality factors in the arrival region has been one of the main factors allowing its expansion. Therefore, this study aimed to collect and identify possible indigenous natural enemies adapting to the new species in the boxwood of the southwest Mediterranean region. Later, the efficacy of some of the collected species for controlling C. perspectalis larvae was tested in laboratory conditions. The larval collection was carried out in successive years in the boxwood of the region. Several collected larvae were infected with an entomopathogen, Beauveria bassiana, or parasitized by Compsilura concinnata, both common in native Lepidoptera caterpillars. The B. bassiana strain was found to be highly virulent against the developed larvae of C. perspectalis, which suggests that B. bassiana may be an effective treatment in parks and gardens when the first overwintering larvae are detected. The biology of the parasitoid identified is not very well known in Europe, which suggests the necessity of studying its biology and alternative hosts in the region in order to improve its population

Entomopathogenic fungi associated with the main insect pest in the Northeast of Portugal: preliminary results

Due to the problems caused by the use of chemical insecticides for humans and environment alternative pest control methods are an important topic of research. The use of microbial insecticides especially fungal agents are an attractive and promising alternative for biological control of insect pests. The aim of this work was to identify naturally occurring entomophatogenic fungi on the olive moth, Prays oleae Bern., in the northeast of Portugal, as first step to select biological control agent again this olive pest. The experimental work was carried out during 2007 in the three generation of the insect (phyllophagous, anthophagous and carpophagous generation). In each generation P. oleae larvae and pupae were collected in different groves and were put in glass vials in a climatic chamber with a photoperiod of 12h light:12h dark, 22ºC (light): 16ºC (dark) and 60% relative humidity, until emergency of the adults. From dead larvae, fungi were isolated on PDA plates and incubated at room temperature. Pure cultures were morphological and molecularly identified based on the ITS region of the rDNA. From the identified species Beauveria bassiana Vuill. and Cordyceps sinensis (Berk.) Sacc. were the most promising being B. bassiana the most abundant one.This work was partially financed by the project INTERREG III