Relative quantification of chrysanthemum yellows (16Sr I) phytoplasma in its plant and insect host using real-time polymerase chain reaction

A real-time polymerase chain reaction (PCR) method for the quantification of chrysanthemum yellows (CY) phytoplasma DNA in its plant (Chrysanthemum carinatum) and insect (Macrosteles quadripunctulatus) host is described. The quantity of CY DNA was measured in each run relative to the amount of host DNA in the sample. Primers and a TaqMan probe for the specific PCR amplification of phytoplasma DNA were designed on a cloned CY-specific ribosomal fragment. Primers and TaqMan probes were also designed on sequences of the internal transcribed spacer region of the insect’s ITS1 rDNA and of the plant’s 18S rDNA for amplification from C. carinatum and M. quadripunculatus, respectively. Absolute quantification of CY DNA was achieved by comparison with a dilution series of the plasmid containing a CY 16S rDNA target sequence. Absolute quantification of plant and insect DNAs was achieved by comparison with a dilution series of the corresponding DNAs. Quantification of CY DNA in relation to host DNA was finally expressed as genome units (GU) of phytoplasma DNA per nanogram of host (plant or insect) DNA. Relative quantification avoided influences due to different yields during the DNA extraction procedure. The quantity of CY DNA was about 10,000–20,000 GU/ng of plant DNA and about 30,000–50,000 GU/ng of insect DNA. The method described could be used to phytoplasma multiplication and movement in different plant and insect hosts.     

Potential Non-target Effects of a Biological Control Agent, Prickly Pear Moth, Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), in North America, and Possible Management Actions

Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), the poster child of biological control, has recently invaded the United States. The first US record was at Big Pine Key, Florida, in 1989. Since then it has moved rapidly northward into South Carolina. Fears are high that it will disperse, either on its own, or with human help, into the US desert southwest and Mexico. There are at least 31 species of prickly pear in the US that are likely to be attacked by Cactoblastis and 56 species in Mexico. As well as the threat to wild cacti, there are over 250,000 ha of Opuntia plantations in Mexico that support a thriving agricultural industry, most of which is centered on harvesting fruits or pads. Possible control measures include classical biological control using parasitoids or pathogens from South America, chemical control or F₁ sterility, as has been used successfully for the codling moth. However, most of these options appear to have insurmountable difficulties. Classical biological control raises the fear of further non-target effects of natural enemies on native cactus herbivores. Chemical control has potential non-target effects on other (rare) insects and is expensive. F₁ sterility is also expensive and is not self-sustaining, requiring considerable and continual human input. Nevertheless, research on control options is vital as is further work on the rate of spread and impact of Cactoblastis in the United States Southeast, so that we can be as well prepared as possible to deal with this threat when it arrives in Arizona, California, and Mexico.     

Host Location and Ovipositional Behavior of Platygaster demades Walker (Hymenoptera: Platygastridae), an Egg Parasitoid of Apple and Pear Leaf Curling Midges

The foraging responses of 1–2-day-old naïve female Platygaster demades to odors of apple and pear foliage and host insect eggs were measured. The host origin of P. demades had no effect on the parasitoids’ longevity, host preference, or foraging behavior. Four distinct behaviors related to oviposition were identified. In choice experiments, more female parasitoids responded to apple foliage with no midge eggs than to midge eggs alone. In a Y-tube olfactometer, parasitoids preferred the plant cues to clean air, and responded equally to both apple and pear odors. The results indicate that P. demades utilizes plant cues to locate the habitat of its host and then searches for host eggs to parasitize.     

Status of biological control by egg parasitoids of Diaprepes abbreviatus (Coleoptera: Curculionidae) in citrus in Florida and Puerto Rico

Eggs of Diaprepes abbreviatus (L.) were routinely monitored in citrus groves at ten locations in Florida during 1997 and 1998 to study egg parasitism. One citrus location was studied in Puerto Rico. No native parasitoids were recovered from 1,337 D. abbreviatus egg masses studied in Florida citrus. In contrast, an average of 35.5% (range 12.5 to 68.8%) parasitism of egg masses was reported in Puerto Rico. The parasitoids Aprostocetus gala, Horismenus spp, and Quadrastichus haitiensis were recovered from the eggs of D. abbreviatusfstudied in Puerto Rico. The Horismenus parasitoids were suspected hyperparasitoids. Releases of the parasitoid Ceratogramma etiennei from Guadeloupe were initiated during 1998 at each of the Florida research sites. By the end of 1998, C. etiennei had been recovered from D. abbreviatus eggs at two of nine locations in Florida citrus. The parasitoid was recovered from 1 of 34 egg masses at one of these locations during the month of September and from 3 of 34 egg masses at the other location during the month of November. Whether or not C. etiennei establishes itself at one or more locations in Florida remains to be seen.     

The urban fire ant paradox: native fire ants persist in an urban refuge while invasive fire ants dominate natural habitats

In contrast to the widespread extirpation of native fire ants (Solenopsis geminata) across southern US following the invasion by imported red fire ants (S. invicta), some residential areas of Austin form unexpected refuges for native fire ants. Ironically, these urban environments provide refuges for the native fire ants while adjacent natural habitats have been overrun by invasive fire ants. Resistance to invasive fire ants in these urban areas occurs mainly in older residential properties constructed prior to the S. invicta invasion, while more recent construction has allowed establishment by S. invicta. The invasive ability of S. invicta is often attributed to escape from parasitoids and efficient dispersal of polygyne multiple queen colonies. Here we also show the importance of landscape parameters in the invasion process, where low levels of disturbance and continuous plant cover in older residential areas form possible barriers to colonization. Dense leaf cover (high NDVI) was also found to be associated with native ant refuges. Long term residential land ownership may have resulted in lower recent disturbance levels and increased plant cover that support refuges of native fire ants.     

Entomopathogenic symbiotic bacteria,Xenorhabdus and Photorhabdus of nematodes

Xenorhabdus and Photorhabdus species are entomopathogenic bacteria with a wide insect host range, that belong to the family Enterobacteriaceae. Xenorhabdus and Photorhabdus species symbiotically associate with nematodes of the families Steinernematidae and Heterorhabditidae respectively. The factor(s) determining the symbiotic interaction between nematodes and bacteria are yet to be identified. Xenorhabdus and Photorhabdus species exist in two main phenotypic forms, a phenomenon known as phase variation. The phase I (or primary form) varies from phase II (or secondary form) in certain physiological and morphological characteristics. There is no variation in the DNA integrity of phase I and phase II and this supports epigenetic regulatory mechanism in phase variation. Certain pathogenic determinants such as pili, lipopolysaccharides and toxins contribute to the pathogenicity of Xenorhabdus and Photorhabdus species, and both appear to be equally pathogenic to insects. The observed similarity in their virulence to insect hosts may reflect possible in vivo conversion of phase II to phase I, however the host cellular invasion and virulence is yet to be properly understood. The virulence of Xenorhabdus variants varies among insects apparently due to factors which include the feeding habits of the insects. The molecular mechanism and biological significance of phase variation are presently unknown.     

Molecular biology of Ri-plasmid—A review

Agrobacterium rhizogenes transfers a segment of its plasmid to the plant genome. The transferred DNA contains genes which are involved in the synthesis of plant hormones. These genes express in the plant cell and give rise to rooty-tumors at the infection site. Transgenic plants can be readily regenerated from the rooty-tumors and the transferred DNA is transmitted to progeny plants. High regeneration potential and sustained maintenance of transferred DNA makes the bacterium a suitable vector for plant genetic engineering. DNA sequences homologous to the transferred DNA ofAgrobacterium rhizogenes were detected in some untransformed plant species suggesting a past infection byAgrobacterium rhizogenes during evolution of some genera, notably Nicotiana.     

Assessment of interspecific interactions among parasitoids on the outcome of inoculative biological control of leafminers attacking chrysanthemum

Indigenous natural enemies occur within field grown crops at varying densities dependent upon a variety of other biotic and abiotic parameters. This natural control often does not provide adequate suppression, which results in the application of other pest management solutions including augmentative biological control. When releasing mass-reared natural enemies into a backdrop of existing natural enemy populations, competitive interactions are likely to occur. To assess the influence of these interspecific interactions on the outcome of such biological control practices studies were conducted in a simulated, field cage grown, cut chrysanthemum production system. Competitive interactions of two commercially available parasitoids were studied both in terms of parasitoid-host population dynamics and the impact of interspecific interactions on crop quality at harvest. The parasitoids Diglyphus isaea and Dacnusa sibirica attacking the leafminer Liriomyza langei were used as the model insect system. Both parasitoids are cosmopolitan and are known to occur in many ornamental production areas. Treatment comparisons included single species releases with complimentary releases of both species either simultaneously or with 2-week time lags, as well as a no release control to measure the background effects of natural mortality. Conclusions drawn from results of population-level studies replicated within and among years were that levels of interspecific competition among parasitoid species were undetectable at leafminer densities typical of field-grown ornamental crops (low densities), and thus, the efficacy of one species released into a backdrop of potentially competing parasitoids did not negatively or positively affect the outcome of the augmentative biological control, nor was there a positive outcome; however, crop quality at harvest was influenced.     

An assessment of interspecific competition between two introduced parasitoids of Diaphorina citri (Hemiptera: Liviidae) on caged citrus plants

Two parasitoids attacking nymphs of Asian citrus psyllid,Diaphorina citri Kuwayama (Hemiptera:Liviidae),Tamarixia radiata (Waterston)(Hymenoptera:Eulophi- dae)and Diaphoreneyrtus aligarhensis (Shafee,Alam &Agarwal)(Hymenoptera:Encyrtidae)are being released in California,USA in a classical biological control program. To evaluate the effect of multiple parasitoid species on D.citri mortality,we conducted mesocosm experiments under controlled conditions using a complete block design with 6 treatments (D.cirri nymphs exposed to:no parasitoids;D.aligarhensis or T.radiata alone;D.aligarhensis or T.radiata released first (by 48 h);and both species released simultaneously).Parasitism of D.citri nymphs by T.radiata exceeded 60%and was unchanged when D.aligarhensis were present.Parasitism by D.aligarhensis was greatest when T. radiata was absent (-28%)and was reduced in all treatments with T.radiata present (<3%).D.cirri mortality and parasitoid-related mortality olD.citri was consistent across parasitoid treatments.Laboratory results suggest that competition between D.aligarhensis and T.radiata is asymmetric and favors T.radiata.It may be difficult for D.aligarhensis to contribute significantly to D.cirri biological control where T.radiata is present.However, results reported here suggest that competition between T.radiata and D.aligarhensis is not likely to reduce parasitism by T.radiata or reduce parasitoid-induced mortality of D.citri.     

Foraging Ants as Scavengers on Entomopathogenic Nematode-Killed Insects

Ants were the most apparent invertebrate scavengers observed foraging on entomopathogenic nematode-killed insects (i.e., insect cadavers containing entomopathogenic nematodes and their symbiotic bacteria) in the present study. Workers of the Argentine ant,Linepithema humile(Mayr), scavenged nematode-killed insects on the surface and those buried 2 cm below the soil surface. Ant workers scavenged significantly more steinernematid-killed (60–85%) than heterorhabditid-killed (10–20%) insects. More 4-day-postinfected cadavers (hosts died within 48 h after exposure to nematodes) were scavenged than 10-day-postinfected cadavers. Ten-day-postinfected hosts contained live infective juvenile nematodes therefore ants may serve as phoretic agents. Other ant species, includingVeromessor andrei(Mayr),Pheidole vistanaForel,Formica pacificaFrancoeur, andMonomoriom ergatogynaWheeler, also scavenged nematode-killed insects. These ant species removed or destroyed about 45% of the steinernematid-killed insects. These results suggest that survival of steinernematid nematodes may be more significantly impacted by invertebrate scavengers, especially ants, than that of heterorhabditid nematodes, and placement of steinernematid-killed insects in the field for biological control may be an ineffective release strategy. Because entomopathogenic nematodes kill insects with the help of symbiotic bacteria, we tested the role of these bacterial species in deterring invertebrate scavengers by injecting bacteria (without nematodes) into insects and placing the cadavers in the field. None of the insects killed by the symbiotic bacterium,Photorhabdus luminescens(Thomas and Poinar) fromHeterorhabditis bacteriophoraPoinar, were scavanged, whereas 70% of the insects killed by the symbiotic bacterium,Xenorhabdus nematophilus(Poinar and Thomas) fromSteinernema carpocapsae(Weiser), and 90% of the insects killed byBacillus thuringiensisBerliner were scavenged by the Argentine ant. We conclude thatP. luminescensis responsible for preventing ants from foraging on heterorhabditid-killed hosts.