Seismic sensor-based management of the red palm weevil Rhynchophorus ferrugineus in date palm plantations.

BACKGROUND: The red palm weevil (RPW), Rhynchophorus ferrugineus, is one of the gravest threats to palm trees. The challenge in monitoring RPW primarily arises from the inconspicuous presence of larvae within the stem, which is often devoid of noticeable symptoms. This study looks at the use of seismic sensors in RPW management in commercial date palm plantations. It explores whether the data garnered from the sensor domain, and its translation into the health status of date palms, can reliably inform precise decision-making. RESULTS: Sensor and damage index values, as gauged by the Agrint IoTree seismic sensor, vividly mirrored RPW colonization activity. They also accurately portrayed the impact of three distinct insecticides: imidacloprid, phosphine, and entomopathogenic nematodes. The seismic values and damage index of healthy untreated palms strongly supported the decision to pursue tree recovery. Furthermore, this facilitated the computation of recovery pace discrepancies across the tested treatments, measured as the number of days required for tree restoration. CONCLUSIONS: Our findings underscore the practicality of employing seismic sensors, as exemplified by the IoTree system and its network services, to both monitor and assess palm tree health. Furthermore, it validates their efficacy in evaluating the efficiency of management strategies adopted against RPW, all grounded in a wealth of sensor-derived data. © 2023 Society of Chemical Industry.

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.

Preinvasion Assessment of Exotic Bark Beetle-Vectored Fungi to Detect Tree-Killing Pathogens.

Exotic diseases and pests of trees have caused continental-scale disturbances in forest ecosystems and industries, and their invasions are considered largely unpredictable. We tested the concept of preinvasion assessment of not yet invasive organisms, which enables empirical risk assessment of potential invasion and impact. Our example assesses fungi associated with Old World bark and ambrosia beetles and their potential to impact North American trees. We selected 55 Asian and European scolytine beetle species using host use, economic, and regulatory criteria. We isolated 111 of their most consistent fungal associates and tested their effect on four important southeastern American pine and oak species. Our test dataset found no highly virulent pathogens that should be classified as an imminent threat. Twenty-two fungal species were minor pathogens, which may require context-dependent response for their vectors at North American borders, while most of the tested fungi displayed no significant impact. Our results are significant in three ways; they ease the concerns over multiple overseas fungus vectors suspected of heightened potential risk, they provide a basis for the focus on the prevention of introduction and establishment of species that may be of consequence, and they demonstrate that preinvasion assessment, if scaled up, can support practical risk assessment of exotic pathogens.

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.

Diversity of Bacterial Biota in Capnodis tenebrionis (Coleoptera: Buprestidae) Larvae.

The bacterial biota in larvae of Capnodis tenebrionis, a serious pest of cultivated stone-fruit trees in the West Palearctic, was revealed for the first time using the MiSeq platform. The core bacterial community remained the same in neonates whether upon hatching or grown on peach plants or an artificial diet, suggesting that C. tenebrionis larvae acquire much of their bacterial biome from the parent adult. Reads affiliated with class levels Gammaproteobacteria and Alphaproteobacteria (phylum Proteobacteria ca. 86%), and Actinobacteria (ca. 14%) were highly abundant. Most diverse reads belong to the families Xanthomonadaceae (50%), Methylobacteriaceae (20%), Hyphomicrobiaceae (9%), Micrococcaceae (7%) and Geodermatophilaceae (4.5%). About two-thirds of the reads are affiliated with the genera Lysobacter, Microvirga, Methylobacterium, and Arthrobacter, which encompass species displaying cellulolytic and lipolytic activities. This study provides a foundation for future studies to elucidate the roles of bacterial biota in C. tenebrionis.

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.

Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism.

The mutualism between xyleborine beetles in the genus Euwallacea (Coleoptera: Curculionidae: Scolytinae) and members of the Ambrosia Fusarium Clade (AFC) represents one of 11 known evolutionary origins of fungiculture by ambrosia beetles. Female Euwallacea beetles transport fusarial symbionts in paired mandibular mycangia from their natal gallery to woody hosts where they are cultivated in galleries as a source of food. Native to Asia, several exotic Euwallacea species were introduced into the United States and Israel within the past two decades and they now threaten urban landscapes, forests and avocado production. To assess species limits and to date the evolutionary diversification of the mutualists, we reconstructed the evolutionary histories of key representatives of the Fusarium and Euwallacea clades using maximum parsimony and maximum likelihood methods. Twelve species-level lineages, termed AF 1-12, were identified within the monophyletic AFC and seven among the Fusarium-farming Euwallacea. Bayesian diversification-time estimates placed the origin of the Euwallacea-Fusarium mutualism near the Oligocene-Miocene boundary ∼19-24 Mya. Most Euwallacea spp. appear to be associated with one species of Fusarium, but two species farmed two closely related fusaria. Euwallacea sp. #2 in Miami-Dade County, Florida cultivated Fusarium spp. AF-6 and AF-8 on avocado, and Euwallacea sp. #4 farmed Fusarium ambrosium AF-1 and Fusarium sp. AF-11 on Chinese tea in Sri Lanka. Cophylogenetic analyses indicated that the Euwallacea and Fusarium phylogenies were largely incongruent, apparently due to the beetles switching fusarial symbionts (i.e., host shifts) at least five times during the evolution of this mutualism. Three cospeciation events between Euwallacea and their AFC symbionts were detected, but randomization tests failed to reject the null hypothesis that the putative parallel cladogenesis is a stochastic pattern. Lastly, two collections of Euwallacea sp. #2 from Miami-Dade County, Florida shared an identical cytochrome oxidase subunit 1 (CO1) allele with Euwallacea validus, suggesting introgressive hybridization between these species and/or pseudogenous nature of this marker. Results of the present study highlight the importance of understanding the potential for and frequency of host-switching between Euwallacea and members of the AFC, and that these shifts may bring together more aggressive and virulent combinations of these invasive mutualists.

Paternal inheritance in mealybugs (Hemiptera: Coccoidea: Pseudococcidae).

Mealybugs have a haplodiploid reproduction system, with paternal genome elimination (PGE); the males are diploid soon after fertilization, but during embryogenesis, the male paternal set of chromosomes becomes heterochromatic (HC) and therefore inactive. Previous studies have suggested that paternal genes can be passed on from mealybug males to their sons, but not necessarily by any son, to the next generation. We employed crosses between two mealybug species--Planococcus ficus (Signoret) and Planococcus citri (Risso)--and between two populations of P. ficus, which differ in their mode of pheromone attraction, in order to demonstrate paternal inheritance from males to F2 through F1 male hybrids. Two traits were monitored through three generations: mode of male pheromone attraction (pherotype) and sequences of the internal transcribed spacer 2 (ITS2) gene segment (genotype). Our results demonstrate that paternal inheritance in mealybugs can occur from males to their F2 offspring, through F1 males (paternal line). F2 backcrossed hybrid males expressed paternal pherotypes and ITS2 genotypes although their mother originated through a maternal population. Further results revealed other, hitherto unknown, aspects of inheritance in mealybugs, such as that hybridization between the two species caused absence of paternal traits in F2 hybrid females produced by F1 hybrid females. Furthermore, hybridization between the two species raised the question of whether unattracted males have any role in the interactions between P. ficus and P. citri.

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.

Evidence for gene flow between two sympatric mealybug species (Insecta; Coccoidea; Pseudococcidae).

Occurrence of inter-species hybrids in natural populations might be evidence of gene flow between species. In the present study we found evidence of gene flow between two sympatric, genetically related scale insect species--the citrus mealybug Planococcus citri (Risso) and the vine mealybug Planococcus ficus (Signoret). These species can be distinguished by morphological, behavioral, and molecular traits. We employed the sex pheromones of the two respective species to study their different patterns of male attraction. We also used nuclear ITS2 (internal transcribed spacer 2) and mitochondrial COI (Cytochrome c oxidase sub unit 1) DNA sequences to characterize populations of the two species, in order to demonstrate the outcome of a possible gene flow between feral populations of the two species. Our results showed attraction to P. ficus pheromones of all tested populations of P. citri males but not vice versa. Furthermore, ITS2 sequences revealed the presence of 'hybrid females' among P. citri populations but not among those of P. ficus. 'hybrid females' from P. citri populations identified as P. citri females according to COI sequences. We offer two hypotheses for these results. 1) The occurrence of phenotypic and genotypic traits of P. ficus in P. citri populations may be attributed to both ancient and contemporary gene flow between their populations; and 2) we cannot rule out that an ancient sympatric speciation by which P. ficus emerged from P. citri might have led to the present situation of shared traits between these species. In light of these findings we also discuss the origin of the studied species and the importance of the pherotype phenomenon as a tool with which to study genetic relationships between congener scale insects.

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.

Molecular evidence of polyandry in the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae).

The occurrence of polyandry in Planococcuscitri, presumed by earlier observations of mating behavior, was confirmed using microsatellite genotyping of pools of over 400 eggs resulting from controlled crosses of one female with two males. The genetic contribution of both mated males was confirmed in 13 out of 43 crosses. In three crosses it was possible to determine that only the first male fertilized the eggs, which may be due to sperm competition or unviable sperm supply. The microsatellite analysis also allowed the confirmation of aspects of the chromosomal inheritance detected previously in cytogenetic studies in Planococcuscitri, namely that only one of the alleles is transmitted by the male, indicating that the males are functionally haploid, supporting the observation of Paternal Genome Elimination (PGE) in these insects.

An inordinate fondness for Fusarium: phylogenetic diversity of fusaria cultivated by ambrosia beetles in the genus Euwallacea on avocado and other plant hosts.

Ambrosia beetle fungiculture represents one of the most ecologically and evolutionarily successful symbioses, as evidenced by the 11 independent origins and 3500 species of ambrosia beetles. Here we document the evolution of a clade within Fusarium associated with ambrosia beetles in the genus Euwallacea (Coleoptera: Scolytinae). Ambrosia Fusarium Clade (AFC) symbionts are unusual in that some are plant pathogens that cause significant damage in naïve natural and cultivated ecosystems, and currently threaten avocado production in the United States, Israel and Australia. Most AFC fusaria produce unusual clavate macroconidia that serve as a putative food source for their insect mutualists. AFC symbionts were abundant in the heads of four Euwallacea spp., which suggests that they are transported within and from the natal gallery in mandibular mycangia. In a four-locus phylogenetic analysis, the AFC was resolved in a strongly supported monophyletic group within the previously described Clade 3 of the Fusarium solani species complex (FSSC). Divergence-time estimates place the origin of the AFC in the early Miocene ∼21.2 Mya, which coincides with the hypothesized adaptive radiation of the Xyleborini. Two strongly supported clades within the AFC (Clades A and B) were identified that include nine species lineages associated with ambrosia beetles, eight with Euwallacea spp. and one reportedly with Xyleborus ferrugineus, and two lineages with no known beetle association. More derived lineages within the AFC showed fixation of the clavate (club-shaped) macroconidial trait, while basal lineages showed a mix of clavate and more typical fusiform macroconidia. AFC lineages consisted mostly of genetically identical individuals associated with specific insect hosts in defined geographic locations, with at least three interspecific hybridization events inferred based on discordant placement in individual gene genealogies and detection of recombinant loci. Overall, these data are consistent with a strong evolutionary trend toward obligate symbiosis coupled with secondary contact and interspecific hybridization.

Morphological and molecular taxonomy of Nidularia balachowskii Bodenheimer (Hemiptera, Coccoidea, Kermesidae) with notes on its life history in Israel.

Descriptions and illustrations of the adult female and first-instar nymph of the kermesid Nidularia balachowskii Bodenheimer, based on the adult female lectotype and paralectotype (here designated), and new material collected from Israel are presented. A key for the identification of first-instar nymphs of Nidularia spp. is offered. Molecular identification of Nidularia balachowskii, using nucleotide sequences from the D2-D3 region of the 28S ribosomal gene, and the mitochondrial Cytochrome Oxidase I (COI) gene, is presented. Morphological and molecular analyses confirm that Nidularia balachowskii is closely related to other species within the Kermesidae. In Israel, this species develops only on Quercus ithaburensis and is univoltine. This is the first detailed report of Nidulariabalachowskii from Israel.

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.

Transgenic organisms expressing genes from Bacillus thuringiensis to combat insect pests.

Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature.

[The pine processionary caterpillar Thaumetopoea pityocampa].

The pine processionary caterpillar Thaumetopoea pityocampa (Lepidoptera: Notodontidae) is considered to be a serious pest of medical importance. The hair on the dorsum of the last instar larvae of the moth may cause urticarial reactions (erucism) as well as eye problems and temporary blindness. In Israel, the pest occurs in all pine plantations as well as on ornamental pine trees in urban areas. The biology, ecology and management of the moth population are discussed as well as the mechanism of action of the urticarial hairs and their medical significance. Awareness of the life cycle and ecology of the pest may reduce the contact of the population with the urticarial hairs and prevent the morbidity caused by it.