Symbiosis and pathogenicity of Geosmithia and Talaromyces spp. associated with the cypress bark beetles Phloeosinus spp. and their parasitoids.

Fungi associated with cypress bark beetles are practically unknown in the Eastern Mediterranean. Our study focused on the fungi associated with the body parts and galleries of two indigenous cypress bark beetles, Phloeosinus armatus and P. bicolor, sampled from Cupressus sempervirens trees in different regions in Israel. Arbitrarily primed PCR, performed on genomic DNA of 302 isolates, clustered the fungal population into five distinct groups. Multilocus phylogeny, split-network analyses and morphological characterization identified the isolates as Geosmithia omnicola, Geosmithia langdonii, Geosmithia sp. 708b, Geosmithia cupressina sp. nov. CBS147103 and Talaromyces cupressi sp. nov. CBS147104. Of these fungal isolates, G. cupressina and T. cupressi are newly described, and their morphological features and phylogenetic designations are presented. Inoculation of intact cypress saplings in an outdoor net-house revealed that only the representative isolate T. cupressi sp. nov. CBS147104 causes 100% disease incidence, whereas Geosmithia spp. isolates are not pathogenic. A number of these fungi were isolated from parasitoids that emerged from branch and stem sections colonized by P. armatus. This study suggests a long and stable association between Phloeosinus and Geosmithia species, and a possible role for additional associated fungal species as pathogens or endophytes of C. sempervirens trees in Israel.

Activity of Metarhizium brunneum and Beauveria bassiana against early developmental stages of the false codling moth Thaumatotibia leucotreta.

This study evaluated the efficacy of two entomopathogenic Hypocrealean fungi, Metarhizium brunneum (laboratory isolate) and Beauveria bassiana (the commercial product Botanigard), for preventative control of the false codling moth, Thaumatotibia leucotreta. The mortality of eggs and first instar larvae was studied in three different assays. First, fungal virulence was examined under optimal laboratory conditions (25 °C, 85% RH) by placing T. leucotreta eggs on conidia-impregnated filter paper. One-day-old eggs and first instar larvae were susceptible to both fungi. In contrast, 5-day-old eggs (advanced embryo development) were susceptible to M. brunneum, but not to B. bassiana. The activity of both fungi against eggs was assessed under two humidity regimes: 85% RH-optimal for fungal germination, and 60% RH-the average humidity in the laboratory. Pieces of parchment paper serving as oviposition surfaces were treated with each of the fungi and introduced to gravid females at different time points after inoculation (0, 2, 7 and 14 days). Although the tested fungal species differed in their virulence to T. leucotreta eggs, both reduced hatching rate under both humidity regimes to 8.3-58.3%, compared to 71.7-83.3% in the control treatments. To evaluate reduction of T. leucotreta infestation of fruit, 'Ori' citrus fruit (easy peeler Citrus sinensis) were treated with each of the fungi. Eggs were placed on the fruit peels and the fruit were maintained under room conditions (25 °C, 60% RH). Between 41.7% and 54.1% of fruit in control groups were infested by the T. leucotreta larvae. Treatments with either of the fungi resulted in about 16% infestation of the fruit with larvae, a marked (3.3-fold) reduction.

Diel periodicity of pheromone release by females of Planococcus citri and Planococcus ficus and the temporal flight activity of their conspecific males.

The diel periodicity of sex pheromone release was monitored in two mealybug species, Planococcus citri and Planococcus ficus (Hemiptera; Pseudococcidae), using sequential SPME/GCMS analysis. A maximal release of 2 ng/h pheromone by 9-12-day-old P. citri females occurred 1-2 h before the beginning of photophase. The highest release of pheromone by P. ficus females was 1-2 ng/2 h of 10-20-day-old females, approximately 2 h after the beginning of photophase. Mating resulted in termination of the pheromone release in both mealybug species. The temporal flight activity of the males was monitored in rearing chambers using pheromone baited delta traps. Males of both P. citri and P. ficus displayed the same flight pattern and began flying at 06:00 hours when the light was turned on, reaching a peak during the first and second hour of the photophase. Our results suggest that other biparental mealybug species display also diel periodicities of maximal pheromone release and response. Direct evaluation of the diel periodicity of the pheromone release by the automatic sequential analysis is convenient and will be very helpful in optimizing the airborne collection and identification of other unknown mealybug pheromones and to study the calling behavior of females. Considering this behavior pattern may help to develop more effective pheromone-based management strategies against mealybugs.

Toxicity assessment of common acaricides and mineral oils on Anagyrus vladimiri, an effective biocontrol agent of citrus mealybug.

Chemical pesticides, while playing an important role in the suppression of insect pests, should be used in a manner that minimizes negative effects on natural enemies. The parasitoid, Anagyrus vladimiri Triapitsyn (Hymenoptera: Encyrtidae), plays an important role in the management of mealybug pests of citrus groves in the Mediterranean region. This study was conducted to evaluate the effect of commonly used acaricides (Spirodiclofen, Spirotetramat, Sulfur, Fenpyroximate, Abamectin) and mineral oils (Levanola, EOS, JMS, and Ultrapaz) on acute mortality of A. vladimiri. Toxicity was assessed in 4 cases: (i) direct spray application on adults, (ii) pesticide application on the mummified host, (iii) feeding with contaminated food, and (iv) contact with pesticide residue. The pesticide Abamectin, applied alone and with Levanola oil was highly toxic to adults in all bioassays, with the exception of direct spray application on the mummified host. Fenpyroximate was found to be highly toxic only when sprayed directly on adults, and sulfur was slightly harmful. Mineral oils were harmful when ingested with food; otherwise, they did not cause appreciable adult mortality. The findings of the present study suggest that all tested materials, with the exception of Abamectin and Fenpyroximate, are compatible with the survival of A. vladimiri. Direct ingestion of oils can, however, cause a degree of mortality. Given that indiscriminate use of these pesticides may affect the population ecology of A. vladimiri, they should be used with caution.

The Dissemination of Metarhizium brunneum Conidia by Females of the Red Palm Weevil, Rhynchophorus ferrugineus, Suggests a New Mechanism for Prevention Practices.

Direct contact between the conidia of entomopathogenic fungi (EPF) and their host is a prerequisite to successful infection; the host can, therefore, be infected by both direct treatment and by transmission of fungal inoculum from infested surfaces. This unique characteristic makes EPF especially relevant for the control of cryptic insects. In the case of the red palm weevil (RPW) Rhynchophorus ferrugineus, the eggs and larvae are almost inaccessible to direct-contact treatment. The objective of the present study was to investigate the mechanism of conidia transmission from a treated surface to host eggs and larvae. Foam pieces infested with Metarhizium brunneum conidial powder, conidial suspension, or distilled water were used as a laying surface for RPW females. The number of eggs laid was not affected by the EPF treatments and ranged from 2 to 14 eggs per female. However, hatching rate and larval survival were significantly reduced in the conidial powder treatment, resulted in 1.5% hatching and no live larvae. In the conidial suspension treatment, 21% of laid eggs hatched, compared to 72% in the control treatment. In both M. brunneum treatments, females' proboscis, front legs and ovipositor were covered with conidia. The females transferred conidia in both treatments to the laying holes, reaching up to 15 mm in depth. This resulted in reduced egg-hatching rate and significant larval mortality due to fungal infection. The stronger effect on egg and larval survival using dry conidia seemed to result from better conidial adhesion to the female weevil in this formulation. In future studies, this dissemination mechanism will be examined as a prevention strategy in date plantations.

Absolute Configuration of the Spherical Mealybug Nipaecoccus viridis Sex Pheromone, γ-Necrodyl Isobutyrate: Chemoenzymatic Synthesis and Bioassays.

The spherical mealybug, Nipaecoccus viridis (Hemiptera: Pseudococcidae), is a major global pest causing feeding damage to leaves and fruits of citrus varieties, soybean, mango, pomegranate, and grapevines. Females of the mealybug release a sex pheromone that was identified recently as a mixture of γ-necrodyl isobutyrate and γ-necrodol. The identification required synthesis based on a natural source of trans-α-necrodol, of unknown chirality, obtained from essential oil of Spanish lavender, Lavandula luisieri. To determine the chirality of the sex pheromone, here, we synthesize the γ-necrodyl acetate enriched in (+)-(S)-enantiomer and separate the enantiomers using a lipase enzyme. We confirm that the natural components, both in the mealybug and in the lavender essential oil, consist of (-)-(R)-enantiomers. Bioassays conducted in the lab and field show that males are attracted to (-)-(R)-γ-necrodyl isobutyrate alone.

Male behaviors reveal multiple pherotypes within vine mealybug Planococcus ficus (Signoret) (Hemiptera; Pseudococcidae) populations.

The vine mealybug (VM) females collected in Israel produce two sex pheromone compounds: lavandulyl senecioate (LS) and (S)-lavandulyl isovalerate (LI). The males display ambiguous behavior to LI: repulsion in the vineyard and attraction of laboratory-reared males. We addressed the question of individual male behavior, i.e., do males respond to both LS and LI, or might they display a distinct response to each of the two pheromone compounds. We compared male pherotype frequencies between wild-caught and laboratory-reared populations. Then, we examined the relationship between pherotype composition and male capture rates in pheromone traps. Finally, we addressed the heredity of the pherotypes. The Israeli VM populations contain nine different male pherotypes, as defined according to the male behavior to pheromone compounds. The studied Portuguese populations included five of the nine pherotypes; none of the Portuguese males were attracted to LI. It seems that the high frequency of males that were attracted to LI is related to dense VM populations. It is hypothesized that selection for the male pherotypes, I males, those that respond to LI, occur under high-density rearing conditions. This may result from shorter development times of males and females that produce more I male pherotypes. The lower relative frequency of trapping of males in LI-baited traps than expected from the percentage determined in a Petri dish arena suggests that males that respond solely to LS (S males) are better fliers. The results also suggest that the pherotype trait is inherited by both sexes of the VM.

Diversity of Ooencyrtus spp. (Hymenoptera: Encyrtidae) parasitizing the eggs of Stenozygum coloratum (Klug) (Hemiptera: Pentatomidae) with description of two new species.

Ooencyrtus spp. (Hymenoptera, Chalcidoidea, Encyrtidae) are important natural enemies of agricultural and forest insect pests, and are distributed worldwide. Their reduced dimensions, highly variable morphological characters and possible effect of wide host range and abiotic factors, make correct identification at the species level particularly difficult. This paper combined molecular, morphological, and biological data to characterize a group of Ooencyrtus spp. emerging from the eggs of the variegated caper bug, Stenozygum coloratum in the east Mediterranean area. COI and ITS2 sequencing revealed the presence of six and five divergent clades, respectively. Three clades were identified as Ooencyrtus telenomicida, Ooencyrtus pityocampae and O. pistaciae. Two clades represent new species which are here described and named Ooencyrtus zoeae and Ooencyrtus mevalbelus. These features were combined with reliable morphological characters to facilitate the separation of these species. A dichotomous key and a new synonymy are proposed. Ooencyrtus pistaciae had two distinct COI clades but only one ITS2 clade. Crossbreeding trials that included Ooencyrtus telenomicida, Ooencyrtus melvabelus sp. nov. and Ooencyrtus zoeae sp. nov. confirmed their reproductive isolation. COI sequences showed 0-0.8% and 4-9% within and between-species genetic differences, respectively. ITS2 showed 0.4-5.9% genetic differences between species, with no genetic differences within species. Haplotype diversity of Israeli and Turkish populations of the various species was 0-0.98 and was particularly low in Ooencyrtus pityocampae, whose Israeli population showed no diversity. The discovery of the Ooencyrtus spp. on the eggs of the caper bug, and their abundance support the idea that the bug can be used as an alternative host for augmentation of populations of these parasitoids in agricultural and forestry systems.

Genetic diversity and host alternation of the egg parasitoid Ooencyrtus pityocampae between the pine processionary moth and the caper bug.

The increased use of molecular tools for species identification in recent decades revealed that each of many apparently generalist parasitoids are actually a complex of morphologically similar congeners, most of which have a rather narrow host range. Ooencyrtus pityocampae (OP), an important egg parasitoid of the pine processionary moth (PPM), is considered a generalist parasitoid. OP emerges from PPM eggs after winter hibernation, mainly in spring and early summer, long before the eggs of the next PPM generation occurs. The occurrence of OP in eggs of the variegated caper bug (CB) Stenozygum coloratum in spring and summer suggests that OP populations alternate seasonally between PPM and CB. However, the identity of OP population on CB eggs seemed uncertain; unlike OP-PPM populations, the former displayed apparently high male/female ratios and lack of attraction to the PPM sex pheromone. We studied the molecular identities of the two populations since the morphological identification of the genus Ooencyrtus, and OP in particular, is difficult. Sequencing of COI and ITS2 DNA fragments and AFLP analysis of individuals from both hosts revealed no apparent differences between the OP-PPM and the OP-CB populations for both the Israeli and the Turkish OPs, which therefore supported the possibility of host alternation. Sequencing data extended our knowledge of the genetic structure of OP populations in the Mediterranean area, and revealed clear separation between East and West Mediterranean populations. The overall level of genetic diversity was rather small, with the Israeli population much less diverse than all others; possible explanations for this finding are discussed. The findings support the possibility of utilizing the CB and other hosts for enhancing biological control of the PPM.

The basis for rootstock resilient to Capnodis species: screening for genes encoding δ-endotoxins from Bacillus thuringiensis.

BACKGROUND: Conventional methods often fail to control the flatheaded borers Capnodis spp., major pests of stone fruit trees; the larvae are protected from insecticides and predation because they feed deep in the roots. A potential solution is transgenic trees producing in their roots toxic compounds such as Cry proteins of Bacillus thuringiensis (Bt). RESULTS: Toxicities against Capnodis larvae were demonstrated by exploiting a recently designed artificial larval diet and an available collection of field isolated Bt. An isolate of Bt tenebrionis (Btt) from commercial bioinsecticide (Novodor) displayed LC50 and LC95 values of 3.2 and 164 mg g(-1) , respectively, against neonates of Capnodis tenebrionis, whereas values of the most toxic field isolate K-7 were 1.9 and 25.6 mg g(-1) respectively. Weights of surviving larvae after 1 month on diets containing low concentrations of K-7 (0.1-1.0 mg g(-1) ) were lower than on Btt or untreated larvae. K-7 was also toxic against larvae of C. cariosa and C. miliaris and found to harbour genes encoding Cry9Ea-like and Cry23Aa/Cry37Aa binary toxins. CONCLUSION: Larvae of Capnodis spp. are susceptible to Bt Cry toxins. Expressing cry genes active against these pests thus seems a feasible solution towards production of transgenic rootstock trees resilient to the pest.