Colony of D. citri

A greenhouse colony of D. citri was established by collecting 10 adult females with orange abdomens from citrus during Mar 2006. The colony was maintained with methods modified from Skelley and Hoy (2004) in a greenhouse at 2032^°C under a 16L:8D photoperiod. Adult D. citri were allowed to oviposit on tender new growth (flush) produced by small potted citrus trees held in mesh cages (61 × 61 × 61 cm). Upon emergence, adult D. citri were collected to initiate another generation or used for evaluation of conidial (spore) pathogenicity in the serial transfer test or for concentration-mortality tests.

Cultivation of Ifr AsCP

Mycosed D. citri that had been exposed to field-collected cadavers were obtained from Jason Meyer. Inoculated insects were held in a sterile 50-mL centrifuge tube (USA Scientific, Ocala, FL) containing a single mature sour orange leaf (Citrus aurantium L.) and a water-soaked cotton ball to maintain RH at approximately 100%. These cultures were held in a growth chamber at 28 ± I^°C under a 16L:8D photoperiod. Spores from the infected insects were used to inoculate 15 × 1.5-cm petri plates (Fisherbrand, Fisher Scientific, Suwanee, GA) containing dilute SDY media (7.5 g Sabouraud dextrose agar, 5 g yeast extract, and 15 g agar/L of water) and maintained in the growth chamber under the same conditions. Spores from these in vitro cultures were harvested 23 d after inoculation by scraping fungal hyphae and spores off the plates with a sterilized spatula and suspending them in 20 mL of autoclaved deionized water. The suspension was filtered through Miracloth (Calbiochem, EMD Biosciences Inc., La Jolla, CA) and spore concentrations were determined with a hemocytometer. New SDY plates were inoculated with 0.5 mL of a 1 × l0⁷/mL spore suspension. In a similar manner, Ifr AsCP was maintained on Malt Extract Agar (MEA) plates (45 g of malt extract agar/L water, MP Biomedicals, Inc., Solon, OH). Plates were sealed with parafilm (Pechiney Plastic Packaging, Menasha, WI) and held in a growth chamber at 28 ± 1^°C under a 16L:8D photoperiod for 23 d.

Serial Transfer of Ifr AsCP on MEA or SDY Media

Every 23 d, spores were harvested from Ifr AsCP grown on SDY and MEA plates and used to inoculate 2 plates each with 0.5 mL of spore suspension at 1 × 10⁷ spores/mL. The pathogenicity of these spores was assessed in newly emerged adult psyllids from the greenhouse colony. Ten adult psyllids (4- to 5-d old) were aspirated from the colony into a 50-mL centrifuge tube, chilled for 10 min on ice and then placed in a 15 × 1.5-cm sterile petri dish and sprayed with 2 mL of spore suspension (1 × 10⁷ spores/mL) that had been harvested from 2 MEA or 2 SDY plates. Psyllids were treated with autoclaved deionized water as a control. Treated psyllids were then individually transferred to 50-mL centrifuge tubes along with a mature sour orange leaf. A cotton ball moistened with tap water and squeezed to remove excess water was placed in the lid. A single Kimwipe (Kimberly-Clark Corp, Roswell, GA) was used to cover the cotton before fastening the lid. The tubes were incubated at 28 ± 1^°C under 16L:8D photoperiod. Psyllids were observed for mortality every 24 h starting 4 d after treatment because Meyer et al. (2008) had shown that mortality did not occur until at least 6 d after exposure to 1 × 10⁷ spores/mL under these conditions.

Mean (standard deviation = SD) percentage germination for transfers 8, 9, and 10 on SDY media was determined by spreading a thin layer of SDY medium on a plain microscope slide (3 × 1 × 1 mm) (Fisher Brand, Fisher Scientific, Pittsburgh, PA). The slide containing the medium was placed into a chamber with 100% RH overnight and then smeared with 100 µL of spore suspension diluted to 1,000 spores/100 µL. The microscope slides were held under 100% RH overnight at 24–25^°C and 100 spores were scored for germination. A total of 5 slides were scored after each transfer and the mean percentage germination was calculated.

Storage of Infected Psyllid Cadavers

Different methods have been used to store fungal isolates, including storing the fungus on agar slants under mineral oil, storing the fungus along with the growing medium under water or mineral oil, freezing at very low temperatures, or storing the fungus on growing medium at room temperature (Dhingra & Sinclair 1995). These methods require an additional step prior to culturing or use of the fungus, which may lead to contamination due to handling or improper sterilization techniques, as well as additional time before the fungus can be used or tested.

To determine if Ifr AsCP could be stored at -74^°C on psyllid cadavers, which would allow us to maintain Ifr AsCP on psyllids without the need to continuously transfer it to new hosts, cadavers were obtained by touching live adults to an infected psyllid with abundant spores and the newly infected psyllids were held in 50-mL centrifuge tubes with a citrus leaf for food at 28 ± 1^°C under a 16L:8D photoperiod for 72 h, after which 100% mortality was observed. After an additional week, the cadavers were evaluated to confirm that they had spores and the tubes then were placed into sealed plastic bags in a -74^°C freezer. Every 2 weeks, 2 cadavers were randomly chosen and thawed to room temperature. Ten adult psyllids that were 4- to 5-d old were collected from the greenhouse colony in a 50-mL centrifuge vial and chilled for 10 min on ice. Two psyllid cadavers were placed in the tube along with 10 chilled psyllids. The vial was tapped several times on a tabletop to ensure the chilled psyllids encountered the cadaver and then the psyllids were held individually in a new 50-mL tube, as described previously. A control also was included, where chilled psyllids were touched with water. The vials were held in a growth chamber at 28 ± 1^°C under 16L:8D photoperiod. This procedure was followed 5 times every 2 weeks and data were recorded as the number of dead psyllids 24, 48, and 72 h after exposure to the cadavers.

Concentration-Mortality and Time-Response Data

Five different concentrations of Ifr AsCP (1 × 10⁴ to 10⁸ spores/mL), plus a water control, were used to conduct a concentration-mortality test at 28 ± 1^°C under a 16L:8D photoperiod. The bioassay was repeated 5 times at 7-10-d intervals. Ifr AsCP spores were scraped off 2 SDY plates that had been inoculated 23 d earlier. A series of plates was inoculated at different times so that each replicate was conducted with spores from plates that were 23 d old. The spore suspension was vortexed in autoclaved deionized water for 1 min and then filtered. Spores were counted and the required concentrations were obtained by serial dilutions. Twenty adult psyllids that were 4- to 5-d old (both sexes) were used for each concentration in each replicate. Psyllids were collected in 50-mL centrifuge tubes and chilled for 10 min on ice and then placed in a petri dish. Psyllids were sprayed with 2 mL of each spore concentration. Then the psyllids were individually transferred to a sterile 50-mL centrifuge tube and maintained as described previously. Psyllids were observed for mortality every 24 h until 10 d after treatment. Data were analyzed by probit analysis in PoloPlus (Le Ora Software 2002–2003).

Evaluation of Infection with Ifr AsCP During Pilot Field Trials

SDY plates (n = 20) that had been inoculated 24 d previously with Ifr AsCP were used to prepare spore suspensions for field application. The spore suspension was prepared as described above. The spore suspension (concentrated) was stored overnight at 4^°C in sterile 50-mL centrifuge tubes and transported to the field in an ice chest containing ice packs. The spore suspension was diluted to 1 × 10⁷ spores/mL (2.7 L) with autoclaved deionized water immediately before spraying trees at the Citrus Research and Education Center (CREC) near Lake Alfred, Florida.

At the CREC, 3 ‘Hamlin’ orange trees, ca. 2–3 m in height and with flush less than 4 cm in length were sprayed with 1 L of autoclaved deionized water/tree as controls. Subsequently, 3 trees were sprayed with 900 mL of 1 × 10⁷ spores/mL/tree. Before spraying the trees, a pretreatment sample of 5 shoots was collected randomly from each tree, and each shoot was placed in a separate clear plastic bag (25.4 × 30.4 cm polyethylene bag, Fisher Scientific, Suwanee, GA) secured with twist ties. The sprays were applied with a battery-operated sprayer (Scorpion sprayer, AgSouth, LLC, Union City, TN). An hour after treatment, a post-treatment sample of 5 shoots per tree was collected from both the Ifr AsCP- and water-treated trees and placed into clear plastic bags secured with twist ties. The bags were stored in an ice chest with ice packs for the trip to Gainesville, Florida.

Immediately after returning to Gainesville, the numbers of psyllid nymphs, adults and/or eggs were recorded and shoot length measured. Insects also were examined for evidence of infection with Ifr AsCP. The shoots were replaced into the bags with 2 moist paper towels to maintain a high RH and stored in a growth chamber at 23 ± 1^°C under a 16L:8D photoperiod. After 5 d, the number of nymphs and adults that were dead or alive was recorded. Dead nymphs and/ or adults recovered from plastic bags were placed on 9.5 × 1.5 cm petri plates containing crystal violet dodine medium (Chase et al. 1986) to observe if any fungal growth occurred. Dead nymphs were evaluated for visible signs of Ifr AsCP.

Foliage samples were randomly collected weekly twice more after the spray and scored as described above. Data were analyzed by ANOVA (SAS 1999) and means were separated with Fisher's least significant difference (LSD) at a 5% level of significance. Weather data were obtained from the weather station at the CREC ( http://fawn.ifas.ufl.edu/scripts/reportrequest.asp), and mean daily temperature, RH, and rainfall were obtained.

A second pilot field trial was conducted with spores that were ‘mass’ produced by Dr. Jarrod Leland (USDA-ARS) and shipped to Gainesville, FL in a dry state in vacuum-sealed plastic bags. Ifr AsCP was grown in a liquid culture for 3 d, and 50 mL of inoculum was added to 1 kg of barley along with 400 mL of sterile water. The barley was kept in a low-oxygen bag at 60% RH, with the bag manipulated daily to prevent clumping. Spores were harvested after 10 d and stored at 2– 4^°C until the trial was conducted. The day before the spray was applied, the dry Ifr AsCP spores were suspended in autoclaved deionized water and vortexed for 1 min. The suspension was then filtered through a double layer of autoclaved Miracloth (Calbiochem, EMD Biosciences, Inc. La Jolla, CA) and spores were counted with a hemocytometer. The spore suspension (concentrated) was stored overnight at 4^°C in sterile 50mL conical centrifuge tubes and transported to field in an ice chest containing ice packs. The spore suspension was diluted to 1 × 10⁷ spores/mL with autoclaved deionized water in the field just before spraying.

The 3-y-old block of trees sprayed in this pilot field trial was located at the Florida Citrus Foundation research grove, in Orange County, near Winter Garden, Florida and consisted of a Minneola cross (LB89), planted 3.8 m apart in the row with rows 6.1 m apart. The 20 trees were ca. 1.2–1.5 m tall, and the 2 treatments were randomly chosen by coin toss. Trees initially contained flush less than 4 cm long with eggs and small psyllid nymphs. Ten trees were treated first with 500 mL of autoclaved deionized water and then 10 trees were treated with 500 mL of 1 × 10⁷ spores/mL/tree with a battery-operated sprayer (Scorpion sprayer, AgSouth, LLC, Union City, TN).

Sampling and analysis of field samples was conducted as described above, except that each shoot was placed into a separate zip-lock plastic bag (Great Value Zip Close bags 17.8 × 20.3 cm, Walmart Stores Inc., Bentonville, AR) with the goal of maintaining higher RH and preventing loss of psyllid nymphs from the bag. In addition, three dead nymphs per shoot or per bag that appeared to be infected with fungus were placed on a petri dish (6.0 × 1.5 cm, Fisher's Scientific, Suwannee, GA) containing SDY media and the identity of the fungus was subsequently confirmed by the polymerase chain reaction (PCR) (Meyer 2007; Meyer et al. 2008). Foliage samples were collected for 4 weeks after the sprays and scored as described above. Data were analyzed by ANOVA and means were separated with Fisher's least significant difference (LSD) at 5% level of significance. Weather data were obtained from the weather station on the site and mean daily temperature, RH, and rainfall were obtained.

Serial Transfer of Ifr AsCP on MEA and SDY Media

Ifr AsCP produced on SDY medium produced significantly more spores than on MEA (Fig. 1). When spore production was evaluated over time, there was no significant difference in spore production on MEA over the 10 transfers, with spore production ranging from 6.1 × 10⁷ to 7.14 × 10⁷ per 10-cm plate (Fig. 1). Spore production on SDY medium was more variable, and appeared to decline over the 10 transfers. Whether this was due to genetic changes in the Ifr AsCP being transferred or due to a decline in the quality of the media used was not determined. Media was prepared fresh for each transfer, but the dry media was stored at room temperature for > 10 months.

Viability of spores was maintained when Ifr AsCP was grown on SDY media. Mean (SD) percentage germination for transfers 8, 9, and 10 were 97 (2.7), 96 (3.9), and 96 (3.5), respectively. Serial transfer for only 10 generations might not be sufficient to show reduced rates of germination, but the production of spores declined for Ifr AsCP grown on the SDY medium.

The colony phenotypes of Ifr AsCP on the 2 media differed consistently throughout the 10 transfers. Mycelia and spores on the SDY medium appeared dense and grayish in color. When the plates were turned over, there was intense yellow pigmentation. By contrast, the mycelia and spores on the MEA plates were less dense, lighter in color, and no pigmentation was seen on the underside of the cultures.

Storage of Frozen Cadavers

After 10 weeks of storage at -74^°C, Ifr AsCP spores on the psyllid cadavers retained a high degree of virulence. All adults that were tested died within 72 h after being exposed to the frozen cadavers. Thus, freezing for up to 10 weeks at -74^°C did not affect the infectivity of the cadavers and this method of storage has the advantage of reducing handling or processing of the fungus to separate it from mineral oil, water, or other storage media. This storage method should reduce the chance of attenuation of Ifr AsCP, because Ifr AsCP can be maintained on psyllid hosts continuously with little labor. When spores were isolated from the cadavers with a sterile needle and streaked on SDY plates, all single colonies had a phenotype typical of Ifr AsCP.

Fig. 1.

Spore production of Ifr AsCP during 10 transfers on MEA and SDY media.

Concentration-Mortality and Time-Response Data

At concentrations of 1 × 10⁷ and 1 × 10⁸ spores/ mL at 28^°C under a 16L:8D photoperiod, the LT₅₀ for D. citri adults was 111 and 102.5 h, respectively (Fig. 2, Table 1). No mortality was observed in the water controls. The LT₉₉ was 167.4 and 174.6 h, respectively, for the 2 spore concentrations. The time-response lines for the 2 concentrations were not different.

A complete concentration-mortality line was calculated for adult psyllid mortality after 192 h (Table 2, Fig. 3). The LC₅₀ value was 6.8 × 10⁵ spores/mL and the LC₉₉ was 2.2 × 10⁸ spores/mL under these laboratory conditions.

Pilot Field Trials

During the first trial, the RH was approximately 42% on the day the sprays were applied, although RH increased later and averaged 6070% (data not shown). Temperature averages ranged from approximately 12–15^°C; only 2 rains occurred during the trial. The mean number of psyllid nymphs was not significantly different on the shoots prior to treatment with water or Ifr AsCP (F = 0.60, df = 1, P = 0.45) or immediately after the sprays were applied (data not shown). The number of nymphs/shoot on the trees treated with water averaged 14.5 and 12.9 immediately before and after treatment, respectively, which was not significantly different (F = 0.10, df = 1, P = 0.75).

Fig. 2.

Time-response curves for adults of D. citri to 2 concentrations of Ifr AsCP at 28 ± 1^°C, 16L:8D photoperiod and ∼100% RH. The lines were generated by Polo-Plus; the individual points indicate the actual mortality caused by the 5 replicates for each concentration.

TABLE 1.

COMPARISON OF TIME TO DEATH (LT) OF ADULT D. CITRI FROM 2 CONCENTRATIONS OF IFR AsCP TESTED AT 28 ± 1^°C, UNDER 16L:8D PHOTOPERIOD AND ∼ 100% RH.

After holding the immediate post-spray sample shoots for 5 d, no fungal infection was observed on any of the dead nymphs or adults, but this could have been due to the low RH in the field at the time of the spray application resulting in low infection rates, or to the low RH in the bags, which were secured with twist ties. All dead nymphs appeared to have starved and dried out, perhaps because the leaves had fallen off the tender young shoots so that adequate food was not available. In most samples, a large proportion (up to 100%) of nymphs and/or adults were missing from the bags, which had been closed with wire twist ties. We concluded that these small insects escaped through small openings where the bags were secured.

One week after the sprays were applied, there were significantly fewer psyllid nymphs (F = 5.13, df = 1, P = 0.03) on the foliaged treated with Ifr AsCP (data not shown), but 2 weeks after sprays were applied, there were no differences in the mean number of psyllid nymphs/shoot in the 2 treatments (F = 0.39, df = 1, P = 0.54). The number of nymphs was reduced in both, probably because the shoots had matured and predation had eliminated some of the nymphs.

TABLE 2.

CONCENTRATION-MORTALITY DATA AFTER 192 H FOR IFR AsCP APPLIED TO ADULTS OF D. CITRI AT 28 ± 1^°C, UNDER 16L:8D PHOTOPERIOD AND ∼ 100% RH.

Fig. 3.

Concentration-mortality curve for adults treated with Ifr AsCP after 192 h at 28 ± 1^°C, under a 16L:8D photoperiod and ∼100% RH. The line was generated by Polo-Plus and the 5 means for each concentation are represented by the individual symbols, some of which are on top of each other.

Prior to spraying the trees, shoot lengths in the Ifr AsCP- and water-treated trees were significantly different (F = 12.04, df = 1, P = 0.002), with an average length of 3.5 cm in the trees to be treated with water and 2.2 cm in the trees to be treated with Ifr AsCP. However, immediately after the sprays were applied, samples were taken again and there were no significant differences (F = 0.15, df = 1, P = 0.71) in shoot length (2.2 and 2.1 cm, respectively), for the water- and Ifr AsCP-treated trees. One week after the sprays were applied, mean shoot length in the trees treated with Ifr AsCP averaged 7.9 cm, while the shoots in the water-treated trees averaged 4.6 cm, a significant difference (F = 17.54, df = 1, P = 0.0004). Two weeks after treatment, shoots from the Ifr AsCPtreated trees again were significantly longer than on the water-treated trees (F = 7.78, df = 1, P = 0.01). Thus, application of Ifr AsCP appeared to result in increased shoot lengths for 2 weeks after treatment, which could have been due to the reduced number of psyllids on the shoots. Unfortunately, because we could not confirm infection of nymphs with Ifr AsCP in the plastic bags, the reductions in the psyllid densities during the course of the experiment could be attributed to one or more factors, including the effects of Ifr AsCP, the production of adults that left the shoots, and predation on nymphs in both control and treatment trees by lady beetles, which were abundant in the block.

The data suggest the fungal spray reduced the number of psyllid nymphs, but the problems in containing the nymphs in the sample bags and confirming that the psyllids were infected with the fungus indicated that our evaluation methods were inadequate. It should be noted that Ifr-AsCP-infected nymphs and adults were not observed in the block while sampling, perhaps because psyllids killed by this fungus do not adhere strongly to leaf surfaces (Meyer 2007; Meyer et al. 2008).

A second spray application was conducted on Aug 9, when the RH averaged ca. 67%. Over the course of this second trial, RH varied but averaged 70–80%. Five rains occurred during the trial and temperature averages ranged from 27–30^°C. During this experiment, the shoot lengths of the trees in the 2 treatments did not differ significantly, except during week 3, when the shoots on the Ifr AsCP-treated trees were significantly longer (F = 5.29, df = 1, P = 0.02).

The mean number of psyllid nymphs on the trees prior to treatment and immediately after treatment were significantly different (F = 4.81, df = 1, P = 0.04; F = 4.54, df = 1, P = 0.04, respectively), with more psyllid nymphs on the trees treated with water. Reasons for the pre-treatment differences are unknown. One week after treatment, there were no differences in nymphal psyllid densities between the 2 treatments (F = 0.01, df = 1, P = 0.90), but during the following week, there were fewer (F = 5.40, df = 1, P = 0.02) psyllid nymphs/shoot in the Ifr AsCP-treated trees (mean = 0.08) compared to the water-treated trees (mean = 0.45). By the third week after sprays were applied, there were no significant differences in nymphal psyllid densities, and densities were very low (mean = 0.03 and 0 for the water- and Ifr AsCP-treated trees, respectively). Thus, it was difficult to determine if nymphal psyllid densities declined due to treatment with Ifr AsCP based on the counts.

However, a total of 437 nymphs from the trees treated with Ifr AsCP that were held in the zip-lock plastic bags in the laboratory to determine if they were infected with Ifr AsCP showed substantial levels of infection under laboratory conditions. At least 327 (75%) were visibly infected with a fungus that was morphologically similar to Ifr AsCP. To confirm that the fungus was Ifr AsCP, Meyer (2007) tested 9 randomly selected nymphs with Ifr AsCP-specific PCR primers and all were positive, indicating that Ifr AsCP had persisted in the plot for 3 weeks and had killed the majority of these psyllid nymphs. A total of 731 psyllid nymphs from the trees treated with water were held under the same conditions and 692 were recovered, with only 39 missing from the bags. Of the 692 nymphs recovered, 28 were dead but these were not infected with a fungus. Thus, the zip-lock plastic bags were well suited to containing the psyllid nymphs and the RH was sufficient that visible symptoms could be observed; fewer than 2% of the nymphs on shoots placed on the bags were missing when scored for infection. This suggests that the fungus did kill psyllid nymphs in the block, but that mortality from other natural enemies (especially predators such as coccinellids, which were abundant in the block) reduced psyllid densities in the watertreated trees.