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Diaphorina citri (Hemiptera: Psyllidae) infection and dissemination of the entomopathogenic fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) under laboratory conditions.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), was first discovered in Florida in 1998 and has since dispersed rapidly throughout the state (Halbert & Manjunath 2004). The insect has a narrow host range consisting of plants in the family Rutaceae, including citrus and citrus relatives such as orange jasmine, Murraya paniculata (L.) Jack (Tsai et al. 2000). Diaphornia citri is a vector of the phloem-limited bacterium Candidatus Liberibacter asiaticus, which is always associated with citrus- huanglongbing (HLB HLB Hong Leong Bank
HLB Hydrophilic-Lipophilic Balance
HLB Horton Lees Brogden Lighting Design (company with studios in New York, San Francisco, Los Angeles, and Boston)
HLB Hotels Licensing Board (Singapore) 
), commonly referred to as 'citrus greening disease' (Hung et al. 2004; Manjunath et al. 2007). HLB is one of the most serious plant diseases in citrus on a worldwide scale (Bove 2006) and has been reported in Florida (Tsai et al. 2000; FDACS FDACS Florida Department of Agriculture and Consumer Services  2009).

Direct feeding by D. citri nymphs is primarily on new citrus growth or "flush" (Hall & Albrigo 2007) which can result in distorted, reduced growth of new leaf tissue. Probing by the adult psyllid while searching for the best feeding area on a leaf can transmit HLB. Infected citrus trees may only live 5-8 years and produce irregular shaped, bitter, unmarketable fruit (Halbert & Manjunath 2004; Bove 2006). Considering the seriousness of the disease and its -vector, controlling psyllid populations by the use of chemical insecticides, removing confirmed diseased trees and planting disease-free nursery stock are recommended as management strategies for this pathosystem (Childers & Rogers 2005; Brlansky et al. 2006; Rogers et al. 2006). The present paradigm of an intensive insecticidal control program is economically unsustainable for the grower and will likely interfere with biological control programs in Florida citrus (Michaud & Grant 2003; Michaud & Olsen 2004; Hoy 2005; Stansly & Qureshi 2008). Thus, an integrated pest management Integrated Pest Management (IPM), planned program that coordinates economically and environmentally acceptable methods of pest control with the judicious and minimal use of toxic pesticides.  (IPM (1) (Impressions Per Minute) Generally refers to document scanners that scan both sides of the page at the same time. Thus, a scanner that scans at 100 ppm (pages per minute) can provide 200 ipm. See ppm and document scanner. ) strategy is needed to minimize the use of chemical insecticides and to develop sustainable alternatives for managing psyllid populations.

The entomopathogenic fungus, Isaria fumosorosea (Ifr) Wize (= Paecilomyces fumosoroseus) (Hypocreales: Cordycipitaceae), was recently isolated from mycosed D. citri collected from the underside of foliage on orange trees in Polk County, Florida Polk County is a county located in the U.S. state of Florida. The county seat is Bartow, Florida. Its largest city is Lakeland, Florida. The center of population of Florida is located in the town of Lake Wales [1].  (28[degrees]06'295" N, 81[degrees]42'895" W) (Meyer et al. 2008). Presently, 2 Ifr strains are available for research as blastospore formulations in the U.S.A., PFR 97 20% WDG WDG Web Design Group
WDG Water Dispersible Granule
WDG Wet Distillers Grain (cattle feed)
WDG Web Development Group
WDG Watchdog Timer (microcontroller, semiconductor, non-volatile memory) 
[R] (Certis, Columbia, MD, USA) and Ifr 3581 from the USDA/ARS, NCAUR NCAUR National Center for Agricultural Utilization Research , Peoria, IL, USA (Jackson et al. 1997). Ifr has several characteristics that favor its further evaluation for controlling D. citri; it is native to Florida, can infect a wide range of citrus pests, and is compatible with non-target arthropods (Sterk et al. 1995a, b; Avery 2002; Avery et al. 2008).

Growing concerns about the negative effects of chemical insecticides on workers, food supply, and the environment make microbial microbial

pertaining to or emanating from a microbe.


microbial digestion
the breakdown of organic material, especially feedstuffs, by microbial organisms.
 control of arthropod arthropod

Any member of the largest phylum, Arthropoda, in the animal kingdom. Arthropoda consists of more than one million known invertebrate species in four subphyla: Uniramia (five classes, including insects), Chelicerata (three classes, including arachnids and horseshoe
 pests of tree fruit crops an attractive alternative (Puterka 1999; Subandiyah et al. 2000; Slininger et al. 2003; Dolinski & Lacey 2007; Lacey & Shapiro-Ilan 2008). The most common fungal pathogen application technique, spraying trees with conidial suspensions, can become cost prohibitive for multiple treatments of groves. Therefore, development of a low-cost autodissemination technique for entomopathogenic fungi where the insect can spread the fungus via horizontal transmission horizontal transmission
n.
Transmission of infection by contact.


horizontal transmission Epidemiology The transmission of an infection from one to another person of the same generation in the same population.
 to conspecifics (e.g., during mating) is warranted. Similar autodissemination techniques for controlling pests have been evaluated in other systems (Maniania 2002; Dowd & Vega 2003; Tsutsumi et al. 2003; Scholte et al. 2004; Maniania et al. 2006).

Adult psyllids are attracted to yellow sticky cards in the field (Hall & Albrigo 2007; Hall et al. 2007, 2008; Hall 2009); therefore, it was hypothesized that yellow tags (non-sticky artificial attractant attractant

a material used to attract animals for capture purposes.
) sprayed with Ifr blastospores could potentially be used to horizontally spread the fungus by acquisition and dissemination to other leaves and psyllids in the field. The objectives were (1) to compare the efficacy of yellow tags and leaves sprayed with Ifr blastospores for infecting and colonizing the psyllid and (2) to assess the horizontal transfer of blastospores by the movement of the adult psyllids under laboratory conditions. A Fungal Development Index (FDI FDI

See: Foreign direct investment
), similar to that of Avery et al. (2004), was designed to assess the effect and development of Ifr dosages on the post-lethal period of infected adult psyllids.

MATERIALS AND METHODS

Source of Insects

The USDA-ARS laboratory colony of D. citri was established during early 2000 at the U.S. Horticultural Research Laboratory, Fort Pierce, FL. Originally collected from citrus, the psyllids have been continuously reared on orange jasmine, Murraya paniculata (L.) Jack housed in Plexiglas (0.6 x 0.6 x 0.6 m) or BugDorm-2 cages (MegaView Science Education Services Co., Ltd., Taichung, Taiwan). Original colony has not had field collected psyllids added since establishment.

Citrus Leaves

Duncan grapefruit (Citrus paradisi Macf.) seedlings were grown in Premier Pro-mix General Purpose Growing Medium from seed in size C10 "Cone-tainers"[TM] (Stuewe & Sons, Inc., Corvalis, OR) for approximately 6 months. Detached leaves of similar age and size were washed with water and placed in a fume in ill temper, esp. from impatience.

See also: Fume
 hood to air dry.

Fungal Blastospore Preparation, Deposition, and Viability

A fungal, dessication-tolerant, blastospore-diatomaceous earth formulation of Ifr ARSEF strain 3581, supplied as a powder in vacuum packed 10-g bags was produced and stabilized as previously described (Jackson et al. 2003) and stored at 4[degrees]C. The blastospore suspension was prepared by mixing 2 g of the powder in 100 mL of sterile distilled water, stirring the suspension with a magnetic bar for 30 min and then allowing the diatomaceous earth diatomaceous earth: see diatom.
diatomaceous earth
 or kieselguhr

Light-coloured, porous, and friable sedimentary rock composed of the frustrules (silicate cell walls) of diatoms.
 to settle from the suspension for an additional 30 min. The suspension (50 mL) was then pipetted to a Nalgene[R] aerosol sprayer (Nalge Nunc International, Rochester, NY). Two aliquots were taken prior to spraying from the suspension and the concentration of Ifr blastospores/mL was determined with a hemacytometer hemacytometer /hema·cy·tom·e·ter/ (he?mah-si-tom´e-ter) an apparatus used for making manual blood counts with a counting chamber.

he·ma·cy·tom·e·ter
n.
See hemocytometer.
.

To determine the deposition of Ifr blastospores/ [mm.sup.2], 12 plastic microscope cover slips (Fisher-brand[R] 22 x 22 mm, Fisher Scientific, Pittsburgh, PA) were placed randomly on paper among the leaf sections and yellow plastic tags and sprayed simultaneously and in an identical fashion. The cover slips were allowed to dry for 30 min in a fume hood, then placed upside down on a glass microscope slide in a 50-[micro]L drop of acid fuschin stain. Blastospore density was assessed with a compound light microscope (400X) and a 10 mm reticle ret·i·cle  
n.
A grid or pattern placed in the eyepiece of an optical instrument, used to establish scale or position.



[Latin r
 grid (Hunt Optic and Imaging, Pittsburg, PA).

The viability of blastospores were assessed with 2 potato dextrose dextrose: see glucose.  agar plates sprayed at a rate of 6.0 x [10.sup.7] blastospores/mL. After the plates had been incubated for 12 h at 25 [+ or -] 1.0[degrees]C, 100% RH, the percent viability was determined by viewing a total of 200 blastospores. Blastospores were considered to have germinated if a germ tube had formed. This procedure was repeated for each repetition of the experiment, and the mean percent viability was 85 [+ or -] 8.3%.

Bioassay Petri Dish pe·tri dish
n.
A shallow circular dish with a loose-fitting cover, used to culture bacteria or other microorganisms.



Petri dish

a shallow, circular, glass or disposable plastic dish used to grow bacteria on solid media such as agar.
 Chambers

Petri dishes (100 mm x 15 mm) were lined with filter paper and moistened with 800 [micro]L sterile distilled water. To prepare the leaf sections, each leaf was cut 2.5 cm from the tip across the midrib. The adaxial side of leaves of similar size and top side of yellow plastic tags (Xpress Tags, Brooklyn, NY), were cut to mimic the shape and surface area (range: 101-125 [mm.sup.2]) of the leaf section. These sections were sprayed until runoff with a Nalgene[R] aerosol sprayer held at approximately a 45[degrees] angle. The spray was either sterile distilled water (DW) or an Ifr blastospore suspension (6.0 x [10.sup.7] blastospores/mL) in sterile water. Sprayed leaves and yellow tags were air dried for 30 min.

Bioassay leaf section treatments inside the Petri dish consisted of 4 sections total, with 1, 2, 3 or 4 leaf section(s) sprayed with Ifr. The yellow tag treatments consisted of 3 leaf sections (sprayed with water) and 1 yellow tag (sprayed with either Ifr or water) placed on moistened filter paper. Leaf section treatment combinations were arranged in the following ratios of fungus (blastospores) to distilled water (Ifr to DW): 0:4 (control), 1:3, 2:2, 3:1, 4:0, and yellow tag treatment combinations 1:3 and 0:4 (control). Treatment combinations were oriented in a cross pattern with the leaf section or yellow tag tip pointed toward the center of the dish prior to introducing an adult psyllid inside the Petri dish.

A single (<1 week old) adult psyllid (sex not identified) was allowed to walk on the inside of the Petri dish lid. The lid was then turned over, placed over the bottom of the dish and the adult psyllid was allowed free movement. Each dish chamber was sealed with Parafilm[R] and transferred to a Precision 818[R] low temperature fluorescent illuminated incubator (Precision, Winchester, VA, USA). All treatments were maintained at 25 [+ or -] 1.0[degrees]C under a photoperiod photoperiod /pho·to·pe·ri·od/ (fo´to-per?e-od) the period of time per day that an organism is exposed to daylight (or to artificial light).photoperiod´ic

pho·to·pe·ri·od
n.
 of 16:8 (L:D) at approximately 100% RH for 14 d and observed on a daily basis. There were 8 replicate dish chambers for each treatment and the experiment was repeated 4 times.

Determining Ifr Acquisition and Horizontal Transfer by the Psyllid

Leaf sections and yellow tags were treated and arranged inside the dish chambers as described above for all treatments. Two groups of treatments (8 replicates/treatment) were compared, one with the psyllid present, the other without the psyllid present. The group without the presence of an adult psyllid served as a control for assessing spread of the blastospores within dish chambers in the absence of a psyllid. Leaf section treatment combinations were arranged in the following ratios of fungus (blastospores) to distilled water (Ifr to DW): 0:4 and 1:3. The yellow tag treatments were conducted as previously described.

After a pilot study, fungal hyphae hy·pha  
n. pl. hy·phae
Any of the threadlike filaments forming the mycelium of a fungus.



[New Latin, from Greek huph
 from spores transferred by the psyllid were first observed to grow on the leaf surface under high relative humidity relative humidity
n.
The ratio of the amount of water vapor in the air at a specific temperature to the maximum amount that the air could hold at that temperature, expressed as a percentage.
 conditions (Avery, unpublished data; Fig. 1). Therefore, this new finding was used to evaluate the transfer of fungal spores among leaves in the dish chamber. Untreated leaf sections inside the dish chambers were monitored for the presence of Ifr hyphae growing on the whole leaf with a dissecting binocular microscope binocular microscope
n.
A microscope having two eyepieces, one for each eye, so that the object can be viewed with both eyes.
 (40X). Data obtained from replicated experiments after 14 d were used as criteria for determining acquisition and horizontal transfer by the adult psyllid. In cases where the insect died and mycosed on an untreated leaf section, the leaf was recorded as contaminated by Ifr and horizontally transferred by the adult psyllid.

Fungal Development Index (FDI) Assessment

The degree of fungal development of Ifr on psyllid adults was assessed by a Fungal Development Index (FDI; see Table 3 for summary) modified from Avery et al. (2004). The FDI was used as a measure for estimating establishment speed or infection rate of hosts in each treatment. All assays were rated daily until sporulation sporulation /spor·u·la·tion/ (spor?u-la´shun) formation of spores.

spor·u·la·tion
n.
The production or release of spores.



sporulation

formation of spores or sporozoites.
 of Ifr was observed (FDI value 3.0) on the insect host. Each adult was assessed under a dissecting binocular microscope (40X), and the FDI value for the stage of fungal development observed was recorded. The FDI was used to assess the fungal growth of blastospores after infection of the adults until colonization at 25 [+ or -] 1.0[degrees]C and 100% RH.

The FDI values of 0.0-0.5, which represented the beginning of the growth phase and initial germination germination, in a seed, process by which the plant embryo within the seed resumes growth after a period of dormancy and the seedling emerges. The length of dormancy varies; the seed of some plants (e.g.  of the blastospore, were not assessed. An FDI value of 1.0 was assumed once the insect died; however, this value was not recorded until confirmation of Ifr fungal hyphae was first noticed extending from any part of the body or wings. Once the fungus protruded through the exoskeleton exoskeleton /exo·skel·e·ton/ (-skel´e-ton) a hard structure formed on the outside of the body, as a crustacean's shell; in vertebrates, applied to structures produced by the epidermis, as hair, nails, hoofs, teeth, etc.  of the host insect (FDI values 1.5-2.0), the insect would not recover from the infection. Conidiogenesis was represented by FDI values 2.5-3.0. Each adult was scored for 8 d according to the FDI as a replicate and results were expressed as a daily mean value for all adult psyllids in each treatment.

Statistical Analysis

The mean number of days of adult psyllids survival post Ifr leaf section treatment compared with a yellow tag treatment were assessed by ANOVA anova

see analysis of variance.

ANOVA Analysis of variance, see there
 ([alpha] = 0.05) with mean separation by a Tukey's HSD HSD Human Services Department
HSD High Speed Data
HSD Hillsboro School District (Hillsboro, OR)
HSD Hybrid Synergy Drive (Toyota/Lexus)
HSD High School Diploma
HSD Historical Society of Delaware
 test. In order to determine the percent transfer of Ifr blastospores to untreated leaf sections by adult psyllid movement compared to no psyllid present, data were arcsine-transformed and analyzed by ANOVA ([alpha] = 0.05) with mean separation by a Tukey's HSD test. A Ryan-Einot-Gabriel-Welsh Multiple Range Test was used to analyze the effect of increasing the number of treated leaf sections on the development of Ifr on the adult psyllid (after initial mycosis mycosis: see fungal infection.  until colonization; FDI value 3.0) and between the single treated leaf section compared to the yellow tag treatment using the FDI values. A regression analysis was used to determine if the infection rate of 1 treated yellow tag was as high or higher compared to a treated single leaf section against the psyllid over time. If results and trends per treatment were not significantly different between repetitions of the experiment based on an ANOVA ([alpha] = 0.05), then the data were pooled and analyzed. All statistical tests were conducted by PROC (language) PROC - The job control language used in the Pick operating system.

["Exploring the Pick Operating System", J.E. Sisk et al, Hayden 1986].
 GLM GLM Global Language Monitor
GLM Global Marine (stock symbol)
GLM Graduated Length Method (ski instruction)
GLM Good Looking Mom (used in pediatric practices)
GLM God Loves Me
 procedures of SAS (1) (SAS Institute Inc., Cary, NC, www.sas.com) A software company that specializes in data warehousing and decision support software based on the SAS System. Founded in 1976, SAS is one of the world's largest privately held software companies. See SAS System.  (SAS Institute, Cary, NC, USA).

RESULTS

Efficacy of Treatments

All Ifr treatments were effective in inducing mortality in adult psyllids under the laboratory conditions tested. No significant differences in treatment results were observed (F = 0.01; df = 3, 15; P = 0.100) between repetitions of the experiment; therefore, the data over all repetitions were pooled and analyzed. The mean number of Ifr viable blastospores/[mm.sup.2] deposited on the leaf sections was 1,344 [+ or -] 149.7.

The number of days for the fungus to infect and induce mortality in an adult psyllid ranged from 4.9 to 6.1, and no mortality was observed in the control treatment (Table 1). Mortality rates of adults in chambers with an Ifr- treated yellow tag were not significantly different (P > 0.05) than mortality rates of adults in chambers with Ifr-treated leaves. The number of days adult psyllids survived in chambers with 3 Ifr- treated leaf sections was significantly shorter (F = 5.60; df = 4, 155; P < 0.001) compared to those treatments with fewer leaf sections treated. The days the psyllid survived in treatments with 3 or 4 leaf sections sprayed were similar (P > 0.05), 4.9 [+ or -] 0.21 and 5.2 [+ or -] 0.30, respectively.

Ifr Acquisition and Horizontal Transfer by the Psyllid

The acquisition and percent horizontal transfer of blastospores to untreated leaf surfaces (edge or center) is presented in Table 2. Psyllid movement within chambers did not affect the percent horizontal transfer of the blastospores to the edge of the untreated leaf sections in either the leaf section or yellow tag treatments (F = 1.12; df = 2, 95; P = 0.348). However, in both the leaf section and yellow tag treatments, the presence and movement of psyllids enhanced and had a significant positive effect on the acquisition and spread of the fungus to the central part of untreated leaf sections (F = 6.67; df = 2, 95; P < 0.001).

FDI Assessment of Ifr

Adult psyllids began succumbing to the fungus 2 d post release in all Ifr treatments. A 100-percent mortality of the adult psyllids occurred (FDI value 1.0) and all psyllids in treatments with 3-4 leaves sprayed had mycosed (FDI value 1.5) 5 d post release (Table 3). The fungi on the leaf section effectively infected and colonized the adult psyllid, as compared with the controls for the duration of the experiment under these growing conditions. The yellow tag treatment had a similar effect on the Ifr development as compared to the single leaf section treatment. Ifr developed on the adult psyllids exposed to the tag treatment at a similar rate compared with the sprayed leaf section treatments, except 5 d post application where 3-4 leaf section treatments showed a higher rate (F = 1.92; df = 3, 127; P = 0.0009) compared with the 1-2 leaf section treatments. The total percentage psyllid adults colonized (FDI value of 3.0: covered with mycelium mycelium

Mass of branched, tubular filaments (hyphae) of fungi (see fungus) that penetrate soil, wood, and other organic matter. The mycelium makes up the thallus (undifferentiated body) of a typical fungus.
 and conidia co·nid·i·a  
n.
Plural of conidium.
) for all experiments after 8 d post release was 63 [+ or -] 8.7, 55 [+ or -] 9.1, 77 [+ or -] 7.6, 75 [+ or -] 7.8% for 1, 2, 3, 4 leaf section(s) treated and 73 [+ or -] 8.2% for the yellow tag treatments. The final percent mortality was 91 [+ or -] 5.2, 97 [+ or -] 3.2,97 [+ or -] 3.2,97 [+ or -] 3.1% and 100 [+ or -] 0.0%, respectively. Regression analyses between FDI value (Y) and days of exposure to Ifr treatment (X) were similar between the yellow tag treatment (Y = -0.9 + 0.44X; F = 347.0, Pr > F = < 0.0001, [r.sup.2] = 0.59, slope SEM = 0.023, 239 df) and single leaf section treatment (Y = -0.8 + 0.39X; F = 276.2, Pr > F = < 0.0001, [r.sup.2] = 0.52, slope SEM = 0.023, 239 df). These analyses indicated that Ifr blastospores sprayed on either a leaf or card, infected and developed on the adult psyllid at a similar rate over time. No natural mortality of the adult psyllids (controls) occurred until 8 d post release for either the 4 leaf sections (3.2 [+ or -] 3.2%) or 1 yellow tag plus 3 leaf sections (25.8 [+ or -] 8.0%) treated with water.

DISCUSSION

Assessment of Ifr Treatments

All Ifr-sprayed leaf section treatments resulted in a mortality of >95% of the adult psyllids under laboratory conditions after 8 d with 100% mortality on the yellow tag treatments during the same period. In addition, fungal development of Ifr on psyllids in the yellow tag treatment was similar to the single leaf section treatment, and comparable to the other leaf section treatments. This indicates that psyllids were attracted to the artificial yellow tag and then able to acquire and disseminate the blastospores to the surface of other untreated leaves. Some of the untreated leaf section edges may have become contaminated with the blastospores by mechanical transfer while in the Petri dishes.

Under these optimum growing conditions in the dish chambers, fungal hyphae were observed to grow on both the leaf (edge and center) and plastic tag surface. Lopez-Llorca et al. (1999) observed that I. farinosa first grew on the edges of the leaves and then colonized the palm leaf surface. This is the first report of Ifr producing hyphal extensions on either a leaf or an artificial surface (yellow tag) directly from Ifr blastospores (Fig. 1).

Moribund psyllids that were attached by mycelium to the filter paper, Petri dish or leaf section had succumbed to the fungal infection after they had walked around and contaminated the untreated leaf surfaces. Under high humidity (RH > 80%) some insects would mycose and form a sporulating cadaver cadaver /ca·dav·er/ (kah-dav´er) a dead body; generally applied to a human body preserved for anatomical study.cadav´ericcadav´erous

ca·dav·er
n.
 cemented in a feeding position to any surface by hyphae growing from their tarsi tar·sus  
n. pl. tar·si
1.
a. The section of the vertebrate foot between the leg and the metatarsus.

b. The bones making up this section, especially the seven small bones of the human ankle.

2.
. Similarly, Meyer et al. (2008) observed that moribund adult psyllids were lightly fastened to the leaf or to the side of a centrifuge centrifuge (sĕn`trəfyj), device using centrifugal force to separate two or more substances of different density, e.g., two liquids or a liquid and a solid.  tube by white mycelium of Ifr AsCP emerging from the tarsi. In addition, Ifr hyphae were observed to spread outwards from the cadavers and contaminate the surrounding leaf surface. On plant leaves, Ifr has been observed to colonize col·o·nize  
v. col·o·nized, col·o·niz·ing, col·o·niz·es

v.tr.
1. To form or establish a colony or colonies in.

2. To migrate to and settle in; occupy as a colony.

3.
 several millimeters across the leaf surface and infect aleyrodids (Wraight et al. 1998). Avery (2002) noted that the Ifr hyphae grew 21 mm across a simulated leaf surface to colonize other susceptible greenhouse whitefly pharate adults.

[FIGURE 1 OMITTED]

FDI Assessment

In all treatments, 83% of the adult psyllids were colonized and sporulating (FDI value: 2.5-3.0) by d 8. Infected insects (FDI value: 1.5) were alive and had fungal hyphae protruding from their leg joints immediately prior to mortality, similar to effects observed by Meyer et al. (2008). After psyllids succumbed to fungal infections, fungal development of Ifr progressed to an FDI value of 2.0 or higher the following day under continuous high (RH > 80%) humidity conditions.

Ifr infection rate on the adult psyllids was comparable to that recorded for the greenhouse whitefly maintained under similar laboratory conditions (Avery et al. 2004). All whitefly pharate adults were completely colonized (FDI value of 3.0) in 8 d following topical application and infection with Ifr blastospores under a 16 hr photophase and high relative humidity. Similarly, in sprayed leaf section treatments, over 97% of the adult psyllids were colonized in 8 d, while 100% of the psyllids were colonized in the yellow tag treatments. Overall, our data supported that a yellow card impregnated with blastospores is as effective in contaminating and killing the adult psyllid as spraying several leaf sections under laboratory conditions. However, the efficacy of Ifr for managing the psyllids by either spraying trees or by using yellow cards contaminated with fungal spores requires evaluation under field conditions. In addition, evaluation of the most suitable material for retaining the blastospores on cards in the field also warrants further investigation.

In autodissemination strategies, the ability of insects to acquire and horizontally transfer viable spores is vital to the effectiveness and ultimate success of a fungal biocontrol bi·o·con·trol  
n.
See biological control.



biocontrol  

See biological control.
 pest management program (Roy et al. 2001; Dowd &Vega 2003; Tsutsumi et al. 2003; Scholte et al. 2004; Maniania et al. 2006). The increase in the amount of viable inoculum inoculum /in·oc·u·lum/ (-ok´u-lum) pl. inoc´ula   material used in inoculation.

in·oc·u·lum
n. pl.
 on the leaf surface appears to positively correlate with the rate of acquisition and concomitant increase in mortality of the psyllid. For instance in Table 3 on d 5, as the concentration of Ifr inoculum

increased among leaf sections per dish chamber from 1 leaf section to 3 leaf sections, the host infection and fungal development rate also increased 7 times. Bailey et al. (2007) found that when the Microsphaeropsis ochracea was increased in concentration per leaf surface, the host infection rate also increased. In contrast, Ugine et al. (2005) noted an inverse relationship between acquisition rate (conidia acquired/total conidia applied) and residue concentration of Beauueria bassiana by western flower thrips thrips, minute, agile insects of the order Thysanoptera. Thrips have piercing-and-sucking mouthparts and cup-shaped feet from which bladderlike adhesive organs may be extended. Some species are wingless, but many have four narrow, featherlike wings fringed with hairs. . This size ratio concept is very important when designing an auto-dissemination system with entomopathogenic fungi and warrants further research.

A high incidence of Ifr hyphae being observed along the edges of some non-treated leaves without psyllids present during the experiments was noted (Table 2), which could be attributed at least partially to the edges of these non-treated leaves coming into accidental contact with the edge of a treated leaf. This scenario could be avoided in future studies by fixing the leaf sections to the filter paper. However, the significant increases in Ifr hyphae growing within the center of leaves and yellow tags was attributed to active dissemination by the adult psyllids. Regardless of whether the sprayed surface was an authentic leaf or an artificial attractant tag, psyllid movement caused significant contamination of unsprayed leaf section centers. Meyling et al. (2006) found that insects living in nettle nettle, common name for the Urticaceae, a family of fibrous herbs, small shrubs, and trees found chiefly in the tropics and subtropics. Several genera of nettles are covered with small stinging hairs that on contact emit an irritant (formic acid) which produces a  plants could help spread and disperse B. bassiana from one site to another. In the field, the transfer of Ifr to leaves or flush where psyllids congregate could potentially lead to secondary infection. In a preliminary bottle cage experiment, it was observed that an entire psyllid population living on a citrus seedling became infected after several days exposure to a yellow tag sprayed with Ifr blastospores (Avery, unpublished data). Also, in a pilot field trial (Avery et al. 2009), 33-50% of psyllid eggs and 29-50% nymphs on citrus flush were found infected with Ifr 10-21 d post-spray, respectively. In addition, 100% (3/3) of the adult psyllids caught per yellow card were contaminated and infected with Ifr 28 d post-spray (Avery et al. 2009).

The autodissemination system using a yellow tag contaminated with Ifr blastospores has potential; however, there are many parameters that need to be investigated further in order to determine the efficacy of this strategy for managing psyllid populations in a citrus grove. The efficacy of a yellow tag contaminated with Ifr blastospores as a source for D. citri to spread the fungus to young citrus plants and other psyllids is presently being tested in cages (Moran et al. 2009); if results are promising then this autodissemination strategy will be evaluated in Texas door-yard citrus. However, persistence and viability of the Ifr blastospores on the yellow tag or leaf surface over time under field conditions will help determine the cost effectiveness of such a pest management strategy. To increase the persistence, viability and efficacy of the fungal blastospores, perhaps an adjuvant adjuvant /ad·ju·vant/ (aj?dbobr-vant) (a-joo´vant)
1. assisting or aiding.

2. a substance that aids another, such as an auxiliary remedy.

3.
 could be added. Dunlap et al. (2007) indicated that the speed of the Ifr blastospore germination was improved by adding keratin keratin (kĕr`ətĭn), any one of a class of fibrous protein molecules that serve as structural units for various living tissues. The keratins are the major protein components of hair, wool, nails, horn, hoofs, and the quills of feathers.  hydrolysate hydrolysate /hy·drol·y·sate/ (hi-drol´i-sat) any compound produced by hydrolysis.

protein hydrolysate
 and the number of infective propagules was increased as well.

The yellow tag may only attract a few psyllids for dissemination of Ifr into the grove and timing of application will be crucial. In a field study where D. citri populations were monitored with yellow sticky traps, the mean number of adult D. citri per trap decreased significantly during periods of abundant new flush compared to trap captures immediately before and after new flush was present (Rogers, unpublished data). Therefore, the yellow tags will need to be hung prior to the emergence of the new preferred flush depending on the climatic conditions and phenology phe·nol·o·gy  
n.
1. The scientific study of periodic biological phenomena, such as flowering, breeding, and migration, in relation to climatic conditions.

2.
 (usually before Mar and just prior to Aug) by the psyllids to be most effective. However, this autodissemination strategy could be augmented by the addition of an attractant in the future (El-Sayed et al. 2006; Suckling et al. 2007). Recently, Wenninger et al. (2008) provided behavioral evidence for a female-produced volatile sex pheromone pheromone

Any chemical compound secreted by an organism in minute amounts to elicit a particular reaction from other organisms of the same species. Pheromones are widespread among insects and vertebrates (except birds) and are present in some fungi, slime molds, and algae.
 for the adult psyllid. Perhaps this pheromone, once identified and synthesized could be added to the yellow tag to increase the effectiveness of attracting other adult psyllids and increase the dissemination of the Ifr into the grove, irrespective of the presence of flush.

In the field, the transfer of Ifr to leaves or flush where psyllids congregate could potentially lead to secondary infection producing sporulating cadavers and eventually under high humidity conditions an epizootic ep·i·zo·ot·ic
adj.
Affecting a large number of animals at the same time within a particular region or geographic area. Used of a disease.



ep
 effect. In a preliminary caged laboratory experiment, it was observed that an entire psyllid population living on a citrus seedling became infected after 7 d of exposure to a yellow tag sprayed with Ifr blastospores (Avery, unpublished data) Also, in qualitative assays, Meyer (2007) recorded 100% mortality of adult D. citri that were exposed to Pfr AsCP on sporulating psyllid cadavers. The extent of the epizootic effect is dependent upon the density of the insects in the area where the sporulating cadavers are located (Furlong and Pell 2001; Avery 2002; Klinger et al. 2006).

Entomopathogenic fungi, which are efficient in killing soft bodied, sucking-piercing insects, are being investigated in different parts of the world as biocontrol agents for controlling the Asian citrus psyllid (Subandiyah et al. 2000; Pell 2008). However, currently there are no Ifr biopesticides registered for spraying and controlling the psyllid on fruit crops in the USA. Presently, Certis[R] in Maryland, USA, produces a blastospore formulation of Ifr (Pfr-97 20% WDG[R] that should become registered for use by citrus growers in 2010 (Dimock, personal communication).

Blastospores of different entomopathogenic fungi, including Ifr have been used extensively in pest management programs on a worldwide scale (Avery 2002). Ifr blastospores easily can be mass produced in a shake-flask liquid culture medium (Jackson et al. 1997, 2003; Lozano-Contreras et al. 2007) and only require 6-8 h to germinate (Vega et al. 1999). Considering that southern and central Florida experiences high humidity, it seems that the environmental conditions are conducive for the use of this fungal biopesticide as part of an IPM program in managing all stages of the psyllid population.

Biopesticides can be used as an alternative in a spray program to break the cycle of harder chemicals and prevent the development of resistance (Moore 2008a). The use of Ifr is an environmentally friendly alternative that will have minimal effect on non-target beneficial arthropods present in the grove (Sterk et al. 1995a, b) and can be used with other strategies for sustainable pest management (Shah & Pell 2003). For instance, Etienne et al. (2001) reported Tamarixia radiata, a parasitoid par·a·sit·oid  
n.
Any of various insects, such as the ichneumon fly, whose larvae are parasites that eventually kill their hosts.

adj.
Of or relating to a parasitic insect of this kind.
 of the Asian citrus psyllid established in the Guadeloupe Islands, has provided excellent control of the psyllid even in the presence of an entomopathogenic fungus of the psyllid, Hirsutella citriformis. Both H. citriformis and Ifr are types of native entomopathogenic fungi found in the Florida groves (Meyer et al. 2007, 2008), and should be compatible with T. radiata previously released for the control of the psyllid pest. However, the compatibility of Ifr with T. radiata for controlling the psyllids needs to be tested under field conditions. Lastly, biopesticides, such as Ifr, may be used effectively either alone or in rotation with traditional pesticides for added genetic resistance prevention (Er & Gocke 2004; Kantz 2007; Moore 2008b). However, which chemicals sprayed in the field are compatible with Ifr warrants further investigation.

These autodissemination laboratory studies are the first to evaluate the potential for using Ifr against the Asian citrus psyllid, whether sprayed on trees or on an artificial attractant surface. Based on the results, the use of Ifr for managing the citrus psyllid has demonstrated potential and warrants further testing under field conditions. Lastly, because the psyllid is attracted to the yellow color (Hall & Albrigo 2007; Hall et al. 2007; Hall et al. 2008; Hall 2009), the use of yellow cards impregnated with Ifr blastospores as part of an IPM strategy has potential for providing citrus growers with a cost-effective method for managing psyllids.

ACKNOWLEDGMENTS

We thank Matthew Hentz and Kathy Moulton for technical assistance in rearing and providing D. citri and Anna Sara Hill at the USDA USDA,
n.pr See United States Department of Agriculture.
, ARS, US Horticultural Research Laboratory for growing the citrus seedlings for this research project. Thanks to Phyllis Rundell and Eliza Duane at IRREC IRREC Indian River Research and Education Center (Florida)  for assistance in the preparation of the spray trials and evaluation of the treatments. Statistical analysis assistance and suggestions by Dr. P Stoffella at UF-IFAS-IRREC in Ft. Pierce, Florida, were greatly appreciated. Reviews by Drs. A. Arevalo, S. Arthurs, P Stansly, L. Stelinski, E. Wenninger, and V Wekesa provided constructive criticism for improving the manuscript. This project was funded by the following grant: The Direct Grower Assistance: Development and Evaluation of Citrus Grower Psyllid Management Programs 2008 awarded by the Florida Citrus Advanced Technology Program (FCATP08: Control of Citrus Greening, Canker canker, small sore on the inside of the mouth. A canker appears as a shallow, whitish ulcer surrounded by a thin, red area. It is tender, sometimes painful, and may occur singly or as one of a group of sores.  and Emerging Diseases of Citrus).

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PASCO B. AVERY (1), WAYNE B. HUNTER (2), DAVID David, in the Bible
David, d. c.970 B.C., king of ancient Israel (c.1010–970 B.C.), successor of Saul. The Book of First Samuel introduces him as the youngest of eight sons who is anointed king by Samuel to replace Saul, who had been deemed a failure.
 G. HALL (2), MARK A. JACKSON (3), CHARLES A. POWELL (1) AND MICHAEL E. ROGERS (4)

(1) University of Florida, Institute of Food and Agricultural Sciences The University of Florida’s Institute of Food and Agricultural Sciences (UF/IFAS) is a federal-state-county partnership dedicated to developing knowledge in agriculture, human and natural resources, and the life sciences, and enhancing and sustaining the quality of human , Indian River Research and Education Center, 2199 South Rock Road, Fort Pierce, FL 34945, USA

(2) USDA, ARS, U.S. Horticultural Research Laboratory, Subtropical sub·trop·i·cal  
adj.
Of, relating to, or being the geographic areas adjacent to the Tropics.


subtropical
Adjective

of the region lying between the tropics and temperate lands

 Insect Research Unit, 2001 South Rock Road, Ft. Pierce, FL 34945, USA

(3) USDA, ARS, National Center for Agricultural Utilization Research, Crop Bioprotection Research Unit, 1815 North University Street, Peoria, IL 61604, USA

(4) University of Florida, Citrus Research and Education Center, 700 Experimental Station Road, Lake Alfred, FL 33850, USA
TABLE 1. MEAN TIME TO DEATH IN DAYS ([+ or -] SEM) FOR
ADULT PSYLLIDS AFTER RELEASE INTO PETRI
DISH CHAMBERS (a) CONTAINING CITRUS LEAF
SECTION (S) OR YELLOW TAGS SPRAYED WITH
ISARIA FUMOSOROSEA (IFR).

Treatment (a)             Time to death (b) (days)

1 Yellow tag sprayed      5.7 [+ or -] 0.23 (ab)
1 Leaf section sprayed    6.1 [+ or -] 0.37 (b)
2 Leaf sections sprayed   5.9 [+ or -] 0.30 (b)
3 Leaf sections sprayed   4.9 [+ or -] 0.21 (a)
4 Leaf sections sprayed   5.2 [+ or -] 0.30 (ab)

(a) Total number of leaf sections per Petri dish chamber was 4.
The leaf sections and yellow tags were sprayed with blastospores
of Ifr and allowed to dry before introducing a psyllid.
The yellow tag replaced 1 leaf section. No mortality was observed
for the untreated controls (n = 32/treatment).

(b) Mean survival values followed by different letters in a column
are significantly different (Tukey's HSD test, P < 0.001).

TABLE 2. PERCENT HORIZONTAL TRANSFER ([+ or -] SEM) OF BLASTOSPORES
OF ISARIA FUMOSOROSEA (IFR) FROM TREATED TO THE UNTREATED LEAF
SECTION EDGE OR CENTER BY ADULT PSYLLID MOVEMENT IN PETRI DISH
CHAMBERS a HELD AT 25[degrees]C UNDER A 16-H PHOTOPHASE AFTER 14
D.

                                   % Horizontal transfer
                                      [+ or -] SEM (b)

Treatment                Psyllid   Leaf edge

1 Leaf section sprayed   Absent    81.9 [+ or -] 6.4 a (c)
1 Leaf section sprayed   Present   90.3 [+ or -] 4.3 a
1 Yellow tag sprayed     Absent    73.6 [+ or -] 7.0 a
1 Yellow tag sprayed     Present   90.4 [+ or -] 3.1 a

                         % Horizontal transfer
                            [+ or -] SEM (b)

Treatment                Leaf center

1 Leaf section sprayed   23.5 [+ or -] 5.8 a
1 Leaf section sprayed   84.8 [+ or -] 4.9 b
1 Yellow tag sprayed     13.8 [+ or -] 4.0 a
1 Yellow tag sprayed     90.3 [+ or -] 3.7 b

(a) Total number of leaf sections per Petri dish chamber was 4. The
leaf sections and yellow tags were sprayed with blastospores of Ifr
and allowed to dry before introducing a psyllid. The yellow tag
replaced 1 leaf section. No mortality for the control was observed
(n = 24/treatment).

(b) Mean percent horizontal transfer values were arcsine
transformed before being analyzed. Untransformed values followed by
different letters in a column are significantly different (Tukey's
HSD test, P < 0.001).

(c) Mechanical transfer of blastospores from a treated leaf section
or yellow tag to an untreated leaf edge occurred in all
treatments.

TABLE 3. FUNGAL DEVELOPMENT INDEX (FDI) VALUES OF MYCOSIS OBSERVED
DAILY ON ADULT PSYLLIDS INFECTED WITH ISARIA FUMOSOROSEA (IFR)
AFTER EXPOSURE TO SPRAYED CITRUS LEAF SECTION(S) OR A YELLOW TAG IN
PETRI DISH CHAMBERS (a) HELD AT 25[degrees]C UNDER A 16-H
PHOTOPHASE.

                                 FDI values (c) days post release

Treatment (b)                        2                      3

1 Yellow tag sprayed        0.1 [+ or -] 0.01 a    0.1 [+ or -] 0.07 a
1 Leaf section sprayed      0.1 [+ or -] 0.07 a    0.1 [+ or -] 0.07 a
2 Leaf sections sprayed     0.1 [+ or -] 0.01 a    0.1 [+ or -] 0.08 a
3 Leaf sections sprayed     0.1 [+ or -] 0.03 a    0.1 [+ or -] 0.04 a
4 Leaf sections sprayed     0.0 [+ or -] 0.00 a    0.1 [+ or -] 0.08 a
4 Leaf sections untreated   0.0 [+ or -] 0.00 a    0.0 [+ or -] 0.00 a
1 Yellow tag untreated      0.0 [+ or -] 0.00 a    0.0 [+ or -] 0.00 a

                                 FDI values (c) days post release

Treatment (b)                        4                      5

1 Yellow tag sprayed        0.3 [+ or -] 0.14 ab   1.0 [+ or -] 0.23 b
1 Leaf section sprayed      0.3 [+ or -] 0.11 ab   1.0 [+ or -] 0.21 b
2 Leaf sections sprayed     0.4 [+ or -] 0.14 ab   1.1 [+ or -] 0.21 b
3 Leaf sections sprayed     0.6 [+ or -] 0.13 a    1.7 [+ or -] 0.19 a
4 Leaf sections sprayed     0.6 [+ or -] 0.15 a    1.7 [+ or -] 0.19 a
4 Leaf sections untreated   0.0 [+ or -] 0.00 b    0.0 [+ or -] 0.00 c
1 Yellow tag untreated      0.0 [+ or -] 0.00 b    0.0 [+ or -] 0.00 c

                                 FDI values (c) days post release

Treatment (b)                        6                      7

1 Yellow tag sprayed        1.7 [+ or -] 0.23 ab   2.4 [+ or -] 0.15 a
1 Leaf section sprayed      1.6 [+ or -] 0.20 b    2.2 [+ or -] 0.19 a
2 Leaf sections sprayed     1.7 [+ or -] 0.21 ab   2.0 [+ or -] 0.21 a
3 Leaf sections sprayed     2.2 [+ or -] 0.16 a    2.5 [+ or -] 0.17 a
4 Leaf sections sprayed     2.2 [+ or -] 0.18 a    2.5 [+ or -] 0.16 a
4 Leaf sections untreated   0.0 [+ or -] 0.00 c    0.0 [+ or -] 0.00 b
1 Yellow tag untreated      0.0 [+ or -] 0.00 c    0.0 [+ or -] 0.00 b

                              FDI values (c) days
                                 post release

Treatment (b)                          8

1 Yellow tag sprayed        2.8 [+ or -] 0.08 a
1 Leaf section sprayed      2.4 [+ or -] 0.17 a
2 Leaf sections sprayed     2.5 [+ or -] 0.13 a
3 Leaf sections sprayed     2.7 [+ or -] 0.12 a
4 Leaf sections sprayed     2.7 [+ or -] 0.11 a
4 Leaf sections untreated   0.1 [+ or -] 0.05 b (d)
1 Yellow tag untreated      0.1 [+ or -] 0.03 b (d)

(a) Total number of leaf sections per Petri dish chamber was 4. The
leaf sections and yellow tags were sprayed with blastospores of Ifr
and allowed to dry before introducing a psyllid. The yellow tag
replaced 1 leaf section (n = 32/treatment).

(b) FDI values are as follows: 1.0 = insect is dead (whether
infected with Ifr fungus or due to natural mortality); 1.5 =
appearance of Ifr fungal hyphae protruded through the exoskeleton
of the psyllid body; 2.0 = Ifr fungal hyphae protruded through
head, thorax, wings of the psyllid body; 2.5 = Ifr fungal hyphae
protruded through the exoskeleton as in 2.0, plus conidia are first
formed anywhere on the psyllid body; 3.0 = Ifr fungus has colonized
and formed conidia on all sections of the psyllid body.

(c) FDI values in a column followed by the same letter are not
significantly different (REGW multiple range test, P < 0.001).

(d) Natural mortality (FDI = 1.0) 8 d post release for leaf section
and yellow tag treatments were 3.2 [+ or -] 3.2% and 25.8 [+ or -]
8.0%. No mortality was observed due to Ifr infection.
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Author:Avery, Pasco B.; Hunter, Wayne B.; Hall, David G.; Jackson, Mark A.; Powell, Charles A.; Rogers, Mic
Publication:Florida Entomologist
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2009
Words:8079
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