Abundance of Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) neonates falling to the soil under tree canopies in Florida citrus groves.

The purpose of these experiments was to estimate the number and distribution of Diaprepes abbreviatus (L.) neonate larvae dropping from the canopy of infested citrus trees. The number of neonates was monitored in the field using passive funnel traps in two simultaneous experiments and a separate experiment for an additional year. In one experiment, traps were placed from trunk to dripline in the cardinal directions under each of five trees (132 traps total). In a second experiment, eight traps were placed under each tree in the cardinal directions, one trap 30 cm from the trunk and one trap 30 cm from the dripline/direction for 25 trees (200 traps total). Larvae were collected weekly for 50 wk in conical tubes containing ethylene glycol as a preservative. Traps closer to the tree trunk captured more larvae than traps nearer the dripline. The area under the tree canopy was positively correlated with the total estimated number of larvae captured per tree. The estimated number of total larvae/tree over the course of our experiments ranged from 955 to 7,290. The highest number of neonate larvae observed in 1 wk was 67 +/- 6/m2. There was an inverse relationship between the number of traps beneath a tree and the number of trees that needed to be sampled to estimate mean population density with a given precision. However, there was a direct relationship between number of traps/tree and the total number of traps needed for a given precision. This passive technique could be used to quantify the destructive larval stage and to assess D. abbreviatus management strategies.

Incidence of Endemic Entomopathogenic Nematodes Following Application of Steinernema riobrave for Control of Diaprepes abbreviatus.

Control of Diaprepes abbreviatus by endemic and exotic entomopathogenic nematodes (EPN) was monitored during 2000-2001 in two citrus orchards in central Florida (Bartow and Poinciana). Caged sentinel insect larvae were buried beneath citrus trees for 7 days at 1 to 2-month intervals from April to October each year. At Bartow, the survey occurred in experimental plots that were (i) not treated with commercial EPN, (ii) treated twice annually since 1998 with commercially formulated Steinernema riobrave, or (iii) treated twice annually with S. riobrave and liquid fertilization (15 times/year) occurred in place of dry fertilizer (3 times/year) used in the other treatments. Four endemic EPN species, in addition to S. riobrave, were recovered from the sandy soil at Bartow: S. diaprepesi, Heterorhabditis zealandica, H. indica, and H. bacteriophora. Mean insect mortality in control plots was 39.4% (range = 13% to 74%), with seasonal maxima in May to July each year. Endemic EPN were recovered from 55% (range = 22% to 81%) of the cadavers each month. Total numbers of endemic EPN recovered in all plots during 2 years were directly related to the numbers of adult weevils (D. abbreviatus and Pachnaeus litus) captured in modified Tedder's traps and inversely related to recovery of S. riobrave. Insect mortality was higher and cadavers containing endemic EPN were more numerous in untreated control plots than in S. riobrave-treated plots, except during months in which S. riobrave was applied. In treated plots, endemic EPN were recovered from cadavers at twice the rate of S. riobrave. Suppression of endemic EPN in plots treated with S. riobrave, combined with inferior persistence by the introduced species, may have attenuated the net efficacy of S. riobrave against D. abbreviatus. In contrast, H. indica was the only endemic nematode recovered from the sandy clay loam soil at Poinciana, where the average mortality of D. abbreviatus was 12% (range 3% to 20%) and incidence of H. indica did not exceed 8%. Results of these surveys suggest that the regional patterns in the abundance and damage to citrus caused by D. abbreviatus in Florida are regulated by endemic EPN and other soilborne enemies of the weevil.

Phytophthora-Diaprepes Weevil Complex: Phytophthora spp. Relationship with Citrus Rootstocks.

Diaprepes abbreviatus (Coleoptera: Curculionidae) is a root weevil, introduced from the Caribbean Basin into Florida in 1964. The larval stage feeds on fibrous and structural roots of citrus, predisposing the injured root system to infection and girdling by Phytophthora spp. In citrus orchards, the rootstocks trifoliate orange (Poncirus trifoliata) and hybrid 'Swingle' citrumelo (Citrus paradisi × P. trifoliata) are resistant to the complex of P. nicotianae with D. abbreviatus, while 'Cleopatra' mandarin (C. reticulata) is susceptible to this complex. When Phytophthora palmivora is coincident with P. nicotianae in fine-textured, poorly drained soils, Swingle citrumelo is more vulnerable to attack by the complex with P. palmivora than is Cleopatra mandarin. Infestation of 9-month-old seedlings with zero, two, or five neonate larvae resulted in a wide range of fibrous root and taproot damage of trifoliate orange and Cleopatra mandarin. Leakage of reducing sugars increased sharply as injury to the roots exceeded 75%. The relationship between feeding damage and root leakage was similar for the two rootstocks. Thus, reduced root damage was not based on host resistance to larval feeding. Root infection and rhizosphere populations of P. nicotianae were higher on Cleopatra mandarin than on trifoliate orange. Root rot by P. nicotianae did not increase with severity of feeding injury on either rootstock. Root infection and root rot by P. palmivora was more severe on trifoliate orange than on Cleopatra mandarin and increased with severity of larval damage. P. palmivora infected and rotted the taproot of both rootstocks if predisposed by larval feeding, but P. nicotianae did not. Commercial rootstocks are severely damaged by larvae of D. abbreviatus; therefore, tolerance of the Phytophthora-Diaprepes weevil complex should be based on resistance of rootstocks to each Phytophthora sp.

Diel periodicity of emergence of adult citrus rust mites in central Florida.

Emergence of adult citrus rust mites, Phyllocoptruta oleivora (Ashmead) from the imagochrysalis occurred with a diel periodicity that peaked between mid morning and early afternoon. The pattern of emergence did not differ for mites reared from eggs held under different photoperiod regimes. The transition of nymphs to the imagochrysalis stage also showed a diel periodicity, occurring in the evening. The average duration of the imagochrysalis stage at 28 degrees C was 14-15 h. Wetting by rain or dew was simulated by immersing fruit in water each night, but the transition to the imagochrysalis stage or the subsequent emergence pattern of adults was not affected. Adult mites reared from eggs laid during two, consecutive 12-h periods emerged at the same time of day, but the peaks of emergence were separated by 24 h. Eclosion of larvae from eggs laid during two, consecutive 12-h periods did not show a diel periodicity. Eggs continuously immersed in water during their development did not hatch, whereas there was a small, but significant, reduction in the viability of eggs immersed in water each night. These results are discussed in relation to external cues that may entrain the diel patterns of development and adult emergence, and to the adaptive responses of citrus rust mite to cyclic fluctuations of key environmental variables in central Florida.

Hydraulic Lift Increases Herbivory by Diaprepes abbreviatus Larvae and Persistence of Steinernema riobrave in Dry Soil.

Citrus seedlings were grown in double pots that separated the root systems into discrete lower and upper zones to test the hypothesis that hydraulic lift affects persistence and efficacy of entomopathogenic nematodes. Three treatments were established: (i) both pots were irrigated at water potential

Virulence of entomopathogenic nematodes to Diaprepes abbreviatus (Coleoptera: Curculionidae) in the laboratory.

The Diaprepes root weevil, Diaprepes abbreviatus (L.) is the most severe weevil pest in Florida citrus. Entomopathogenic nematodes have effectively suppressed larval populations of D. abbreviatus. Our objective was to conduct a broad laboratory comparison of entomopathogenic nematodes for virulence toward larvae of D. abbreviatus. The study was conducted at three temperatures (20, 24, and 29 degrees C) and included nine entomopathogenic species and 17 strains: Heterorhabditis bacteriophora Poinar (Baine, NJl, Hb, Hbl, HP88, and Lewiston strains), H. indica Poinar, Karunakar & David (original and Homl strains), H. marelatus Liu & Berry (IN and Point Reyes strains), H. megidis Poinar, Jackson & Klein (UK21l strain), H. zealandica Poinar (NZH3 strain), Steinernema riobrave Cabanillas, Poinar & Raulston (355 strain), S. carpocapsae (Weiser) (All strain), S. feltiae (Filipjev) (SN and UK76 strains), and S. glaseri (Steiner) (NJ43 strain). At 20 degrees C, the greatest mortality was caused by S. riobrave although it was not significantly greater than H. bacteriophora (Baine), H. bacteriophora (Hb), H. bacteriophora (Hbl), and H. indica (original). At 24 and 29 degrees C, S. riobrave caused greater larval mortality than other nematodes tested. Two strains of H. indica, H. bacteriophora (Baine), and S. glaseri were next in terms of virulence at 29 degrees C. Our results suggest that S. riobrave has the greatest potential for control of D. abbreviatus.

Eavesdropping on insects hidden in soil and interior structures of plants.

Accelerometer, electret microphone, and piezoelectric disk acoustic systems were evaluated for their potential to detect hidden insect infestations in soil and interior structures of plants. Coleopteran grubs (the scarabaeids Phyllophaga spp. and Cyclocephala spp.) and the curculionids Diaprepes abbreviatus (L.) and Otiorhynchus sulcatus (F.) weighing 50-300 mg were detected easily in the laboratory and in the field except under extremely windy or noisy conditions. Cephus cinctus Norton (Hymenoptera: Cephidae) larvae weighing 1-12 mg could be detected in small pots of wheat in the laboratory by taking moderate precautions to eliminate background noise. Insect sounds could be distinguished from background noises by differences in frequency and temporal patterns, but insects of similarly sized species could not be distinguished easily from each other. Insect activity was highly variable among individuals and species, although D. abbreviatus grubs tended to be more active than those of O. sulcatus. Tests were done to compare acoustically predicted infestations with the contents of soil samples taken at recording sites. Under laboratory or ideal field conditions, active insects within approximately 30 cm were identified with nearly 100% reliability. In field tests under adverse conditions, the reliability decreased to approximately 75%. These results indicate that acoustic systems with vibration sensors have considerable potential as activity monitors in the laboratory and as field tools for rapid, nondestructive scouting and mapping of soil insect populations.

Nitrogen partitioning in Heterorhabditis bacteriophora-infected hosts and the effects of nitrogen on attraction/repulsion.

Entomopathogenic nematode behavior is affected by the condition of their infected hosts. We hypothesized that nitrogen compounds released from infected hosts may be one factor affecting entomopathogenic nematode host-finding and infection behaviors. Our objectives were to (1) investigate the partitioning of nitrogen in Galleria mellonella (L.) infected by Heterorhabditis bacteriophora Poinar and (2) determine attraction and repulsion of H. bacteriophora to various quantities of nitrogen (ammonia). Volatile (ammonia), organic, and inorganic nitrogen forms were monitored during the course of infection. Approximately 0.052 mg of nitrogen was released from a single infected host as volatile ammonia. Most of the ammonia release was detected within the first 3 days postinoculation. Organic nitrogen increased during the course of infection, whereas inorganic nitrogen decreased. The net nitrogen change in the infected host consisted of a loss of approximately 47 mg, most of which was lost within the 1st week of infection. Accelerated loss of nitrogen early in the infection process was likely correlated with activity and growth of bacterial symbionts. Increased organic nitrogen was likely associated with nematode reproduction within the host. Attraction or repulsion of H. bacteriophora to nitrogen (ammonium hydroxide) was measured on agar quadrant plates. Nematodes were attracted to 16 and 160 microg of nitrogen and repelled by concentrations of 1600 and 8000 microg. Our data indicate that nitrogen released from H. bacteriophora-infected hosts attracts nematodes at lower levels (early in the infection) and repels them at higher concentrations (later in the infection process).

Larval growth of Diaprepes abbreviatus (Coleoptera: Curculionidae) and resulting root injury to three citrus varieties in two soil types.

Larval growth and intraspecific competition of Diaprepes abbreviatus (L.) larvae and consequent root injury in container-grown citrus in the greenhouse were evaluated. Roots of Carrizo citrange, Citrus sinesis L. Osbeck x Poncirus trifoliata (L.) Raf.; Cleopatra mandarin, C. reticulata Blanco, and Swingle citrumelo, C. paradisi Macf. x P. trifoliata (L.) Raf. rootstock seedlings grown in Candler fine sand and potting soil were colonized with different populations of D. abbreviatus larvae. Larvae were exposed to the seedlings for 79 d. Larval growth and development increased steadily for approximately 70 d on all rootstock-soil combinations, at which time most larvae were instars 6-8. Most feeding injury occurred to roots when larvae were between instars 3 and 6. Larval weight reached a plateau at approximately 70 d, but often declined between 70 and 79 d. When larvae were small, injury to seedlings developed slowly, primarily on fibrous roots, then feeding increased rapidly, often resulting in total consumption of both fibrous root and bark tissue. Although not statistically significant, root injury developed slightly slower on Swingle citrumelo compared with Carrizo and Cleopatra rootstocks, but damage was comparable by 79 d. Little or no difference in consumptive benefit to the larvae was found between the rootstocks. Based on larval weight days, little feeding injury occurred during the first 21 d, but increased rapidly between 21 and 60 d. Soil type affected the rate of larval growth and development, with potting soil contributing to greater growth rates. Detritus in potting soil provided little or no nutritional resource, suggesting that the effect of potting soil on larval development was primarily physical. In addition, fewer inoculated larvae per seedling exhibited greater weight gains than higher infestation densities, suggesting that intraspecific competition for nutritional resources influenced larval development.

Effects of Culture Method and Formulation on the Virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae).

The Diaprepes root weevil, Diaprepes abbreviatus, is a pest of vegetables, ornamental plants, sugarcane, and citrus in Florida and the Caribbean. The entomopathogenic nematode, Steinernema riobrave, can reduce larval populations of D. abbreviatus substantially. Efficacy of entomopathogenic nematodes, however, may be affected by culture method and formulation. Using D. abbreviatus as the host, we compared the efficacy of two commercial S. riobrave formulations, a liquid and a waterdispersible granule (WDG), with each other and with in vivo produced S. riobrave. Nematodes in the commercial formulations were produced in vitro through liquid fermentation; the in vivo nematodes were cultured in Galleria mellonella and applied in aqueous suspension. Laboratory experiments measured nematode virulence in plastic cups containing soil and seventh-eighth instar D. abbreviatus. One laboratory experiment was conducted using only fresh nematodes (less than 5 days old); another experiment included WDG nematodes that were stored for 25 days at 10 degrees C. Two field experiments were conducted in which nematodes were applied either to potted citrus (containing D. abbreviatus larvae) placed beneath mature citrus trees or to soil directly beneath the tree. In the latter experiment, efficacy was determined by measuring mortality of caged D. abbreviatus larvae that were buried beneath the soil surface prior to application. Mortality of D. abbreviatus treated with nematodes ranged from 80-98% and 50-75% in laboratory and field experiments, respectively. In all experiments, we did not detect any significant effects of culture method or formulation.

Monitoring the susceptibility of citrus rust mite (Acari: Eriophyidae) populations to abamectin.

A citrus leaf disk bioassay was developed to monitor the susceptibility of citrus rust mite, Phyllocoptruta oleivora (Ashmead), populations to abamectin. Disks from leaves of several citrus cultivars were equally suitable bioassay substrates, and there was no difference in mortality when mites were sprayed directly or exposed to dry abamectin residue. The concentration-response relationship was determined at intervals over 2 yr for a reference population of citrus rust mites that had been maintained in culture and never exposed to acaricides. Three diagnostic concentrations of abamectin were selected based on the response of the reference population and were used to test the susceptibility of 15 populations of mites from commercial citrus groves. Comparisons with the reference population showed reduced levels of susceptibility in some populations. Populations of citrus rust mites from 6 commercial groves were sprayed twice in 1997 with combinations of acaricides designed to exert different intensities of selection pressure from abamectin. None of these populations showed a change in their response to abamectin in pre- and postspray bioassays, although their susceptibility was usually less than that of mites from the susceptible reference population. Biweekly counts of rust mites on fruit in these 6 groves suggested that, relative to groves which received no abamectin or 1 abamectin spray, mite control was not adversely affected in the groves sprayed twice with abamectin. The bioassay method is discussed in relation to factors that affect the interpretation of results from its use, and factors that may affect the development of resistance to abamectin in citrus rust mite populations are presented. This study has provided baseline data with which the results of ongoing tests of the response of citrus rust mite populations to abamectin can be compared.

Spatial patterns of entomopathogenic nematodes in microcosms: implications for laboratory experiments.

LABORATORY MICROCOSMS WERE USED TO: i) measure the effects of soil moisture on survival of Steinernema riobravis and ii) investigate the suitability of using microcosms to study motility and survival of these nematodes. Nematodes recovered from soil contained in petri dishes declined by more than 95% during 7 days, whereas nematodes recovered from the inner surfaces of dishes increased 35-fold. After 7 days in dishes, >20 times as many nematodes were recovered from dish surfaces than from soil. Nematodes exhibited a negative geotropism; greater numbers of nematodes were recovered from the lid surfaces than from the surfaces of dishes. Survivorship of nematodes in soil in plastic centrifuge tubes was somewhat greater than in petri dishes, and fewer nematodes ascended above the soil line in tubes than dishes. Downward migration of nematodes was inversely related to soil column diameter, possibly due to relatively unimpeded movement along container surfaces. An assay was developed by which nematodes were rinsed from the inner surfaces of centrifuge tubes into the soil. The resulting slurry was then processed on Baermann trays to recover motile nematodes. Nematode survival in soil in centrifuge tubes was higher at soil moistures between 2-4% than at lower (0.5-1.0%) and higher (4.0-12.0%) moisture levels. Survival of S. riobravis may be enhanced by quiescence induced by moisture deficits.

Estimating Sample Size and Persistence of Entomogenous Nematodes in Sandy Soils and Their Efficacy Against the Larvae of Diaprepes abbreviatus in Florida.

In two studies to estimate sampling requirements for entomogenous nematodes in the field, highest persistence of Heterorhabditis bacteriophora after application occurred beneath the canopies of mature citrus trees. Nematode persistence declined with distance from the center-line of the tree row toward the row-middles. Immediately after nematode application to soil, 32 samples (15 cm deep, 2.5-cm diameter) beneath a single tree were required to derive 95% confidence intervals that were within 40% of mean nematode population density. The estimated probability of measuring the mean density within 40%, using 32 samples, declined to 88% at 2 days post-application and to 76% at 7 days. The persistence in soil of Steinernema carpocapsae, S. riobravis, and two formulations containing H. bacteriophora and their efficacy against the larvae of Diaprepes abbreviatus were compared in a grove of 4-year-old citrus trees. Within 6 days, the recovered population densities of all nematodes declined to <5% of levels on day 0. The recovery of H. bacteriophora during the first 2 weeks was lower than that of the other two species. Steinemema riobravis and both formulations of H. bacteriophora reduced recovery of D. abbreviatus by more than 90% and 50%, respectively. Steinernema carpocapsae did not affect population levels of the insect.

Extraction and characterization of the insecticidal toxin hirsutellin A produced by Hirsutella thompsonii var. thompsonii.

Hirsutellin A (HtA) produced by Hirsutella thompsonii var. thompsonii (strain JAB-04) was extracted and purified using a combination of ion-exchange, gel-permeation, and immunoaffinity chromatography. The identity of the purified HtA was confirmed by amino acid analysis and N-terminal sequencing. Monoclonal antibodies prepared against HtA were capable of detecting 25-50 ng of HtA by direct sandwich ELISA. In addition, utilizing Western blot methods, the antibodies were shown to be specific to HtA. The production of HtA was monitored during submerged fermentation. The peak level of exocellular HtA (13-14 micrograms/ml) was during the late exponential growth phase (39-45 h), determined by utilizing a combination of densitometric analysis of the 16.3-kDa bands on SDS-PAGE gels and ELISA. HtA production was directly correlated with mycelial growth. Twenty-one-hour culture filtrates were highly toxic to larvae of the greater wax moth. Pure HtA at a final concentration of 40 pmol was highly toxic to Galleria mellonella larvae.

Toxicity and pathology of crude broth filtrate produced by Hirsutella thompsonii var. thompsonii in shake culture.

Metabolic products secreted by the fungal mycelia of Hirsutella thompsonii var. thompsonii (CBS 556.77D) in a defined culture broth in shake culture were tested for toxicity to Galleria mellonella larvae and Drosophila melanogaster adults via injection and per os application, respectively. In addition, the toxic effect of broth filtrate was observed in vitro in a cell line of Bombyx mori. Czapek-Dox broth fortified with 1% yeast extract stimulated more rapid mycelial growth and correspondingly more toxin production in time. At 25-30 degrees C, metabolic toxin(s) was detected in broth via bioassay at about 4-5 days postinoculation when mycelial biomass reached 5 mg/ml (dry wt). At these temperatures, biological activity of the filtrate peaked at about 8-10 days when mycelial growth reached a maximum (10 mg/ml, dry wt). This suggests a positive relationship between toxic metabolite and mycelial production. After 10 days, the toxicity of the filtrate appeared to decline gradually. Pathogenicity symptoms of the metabolites developed slowly in both G. mellonella and D. melanogaster. Early signs of lethargy appeared at 4 days postinjection and cumulative mortality of G. mellonella larvae was low after 1 week; however, the percentage of mortality reached 98-100% after 14 days. At death, G. mellonella larvae displayed small dark spots on a brownish cuticle. Histopathological effects were observed in the larval midgut, malpighian tubules, hypodermis, fat body, hemocytes, muscle, and silk glands. Cellular change consisted of pycnosis of the nucleus and a reduction in cytoplasm density. Highest mortality (78.8%) to adult D. melanogaster occurred after 10 days post-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)