Pasco B. Avery

Diaphorina citri (Hemiptera: Psyllidae) Infection and Dissemination of the Entomopathogenic Fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) Under Laboratory Conditions

ABSTRACT The infectivity and horizontal transfer of Isaria fumosorosea Wize among Diaphorina citri Kuwayama was measured using a detached leaf bioassay in which blastospores were sprayed on citrus leaf sections or yellow plastic tags (artificial attractant surface). Four leaf sections or three leaf sections and one yellow tag were placed together in a Petri dish chamber. One to four of the leaf sections or the yellow tag was sprayed with I. fumosorosea (1.2–1.7×103 blastospores/mm2). After treatments dried, a single adult psyllid was released into each chamber. Mortality due to I. fumosorosea for the adult psyllid was observed 4.9 ± 0.21–6.1 ± 0.37 d following exposure to the pathogen. The rate of colonization by I. fumosorosea on adults in chambers with untreated leaf sections and one treated yellow tag was as effective in inducing mortality as in chambers with one treated leaf section at 8 days post application. Under high humidity, I. fumosorosea blastospores readily produced hyphae on the surface of leaves, which was useful for determining if adults were responsible for transmission of the fungus. In chambers with a single treated leaf section, adults came into contact with blastospores and moved these around to the non-treated leaves. The same phenomenon, of psyllid infection and subsequent spreading of the fungus to non-treated leaves, was observed when psyllids were placed into chambers with a treated yellow tag. The use of I. fumosorosea inoculated yellow tags has potential as a psyllid dissemination technique for managing pest populations.

Effects of the fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) on reduced feeding and mortality of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae)

Abstract The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is an important citrus pest primarily because it transmits bacteria putatively responsible for huanglongbing, a serious disease of citrus. We present a study on the effects of blastospore and conidial formulations of Isaria fumosorosea Wize on feeding rates and mortality of adult psyllids in laboratory bioassays. Information on quantities of honeydew droplets was used to make inferences on feeding rates. Psyllids treated with the blastospore formulation of I. fumosorosea produced fewer honeydew droplets compared to the conidial treatment and control beginning within the first 24 h after treatment. The highest daily mean number of droplets thereafter never exceeded 2.4 drops compared to 4 and 8 for the conidial treatment and control, respectively. The mean number (±SEM) of honeydew droplets produced per psyllid per day over 7 days was significantly higher in the control (5.5±0.5) compared to the blastospore treatment; however, there were no significant differences between the treatments. Psyllids treated with the conidial formulation of the pathogen showed no significant reduction in feeding activity until 4 days after treatment. One and 2 days post-exposure, mortality of psyllids in the blastospore treatment ranged from 8 to 25% compared to 0% in the conidial and control treatments. By 7 days post-exposure, psyllid mortality reached 100% under both fungal treatments compared to none in the controls. This study documented that adult psyllids infected by I. fumosorosea (PFR 97) produce less honeydew than healthy psyllids and suggests that they may feed less, which could potentially reduce the spread of huanglongbing.

Broad Spectrum Potential of Isaria fumosorosea Against Insect Pests of Citrus

Renewed interest in the use of entomopatho genic fungi in the management of insect pests led to the evaluation of Isaria fumosorosea Wize (Ifr) (= Paecilomyces fumosoroseus (Wize) A.H.s. Br. & G. Sm.) on citrus pests due to finding the inva sive, Asian citrus psyllid, Diaphorina citri (Ku wayama) (Hemiptera: Psyllidae) infected with a strain of this fungus in a citrus grove (Meyer et al. 2008). The fungus was isolated from mycosed D. citri collected from the underside of foliage on or ange trees in Polk County, Florida. In this study, 3 hemipteran insects (psyllid, leafhopper, aphid) and 1 coleopteran (Diaprepes citrus root weevil) were evaluated for susceptibility to Ifr under lab oratory conditions. The Asian citrus psyllid, D. citri, is the vector of the citrus-infecting bacteria associated with citrus yellow shoot disease, known as Huanglongbing (Bove, 2006; 2008), and the glassy-winged sharpshooter, Homalodisca vitripennis Germar (Hemiptera: Cicadellidae) is a vector for the plant-infecting bacteria, Xylella fas tidiosa which causes citrus variegated chlorosis, scorch-like diseases of fruit crops, and Pierces disease of grapevine (Daugherty & Almeida 2009). the brown citrus aphid, Toxoptera citrici dus (Kirkaldy) (Hemiptera: Aphididae), is a vec tor of citrus tristeza virus, CTV (Roy & Brlansky 2009) and the root weevil, Diaprepes abbreviatus L., is a common pest of citrus which has a broad host range and damages the roots of trees which permits phytopthora infections (Graham et al. 2003). Thus these insect pests of citrus trees were evaluated for susceptibility to strains of the ento mopathogenic fungus, Ifr. Presently, 3 Ifr strains are available for re search as blastospore formulations in the U.S.A. There is the Ifr APOPKA strain, in PFR 97 20% WDG® (Certis, Columbia, Maryland, USA), la beled for controlling psyllids, leafhoppers, and aphids; the Ifr 9901 strain, in NoFlyTM WP (Natu ral Industries, Houston, Texas labeled for control ling whiteflies, aphids and thrips and lastly, the non-commercial strain, Ifr 3581 available from the USDA/ARS, NCAUR, Peoria, Illinois, USA (Jackson et al. 1997). Ifr has several characteris tics that favor evaluation for controlling insect pests of citrus: 1) it is native to Florida, 2) it is compatible with non-target arthropods and 3) it can be horizontally transferred and infect conspe cifics (Sterk et al. 1995a, b; Avery et al. 2008, 2009, 2010). In our experiments only Ifr strain 3581 and PFR 97 were tested on the citrus pest insects. This is the first report indicating that H. vitripennis, T. citricidus and D. abbreviatus were susceptible to Ifr strains under laboratory condi tions.

Evaluation of Isaria fumosorosea (Hypocreales: Cordycipitaceae) for control of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae)

A laboratory bioassay was developed to evaluate strains of Isaria fumosorosea Wize, against Diaphorina citri. Up to 100% of adult psyllids were killed at concentrations between 106 and 107 blastospores/ml after 12 days, with derived LC50 values (at 7 days post treatment) between 1.4 × 105 and 2.0 × 106 blastospores/ml for strains ARSEF 3581, FE 9901 and Apopka-97. A significantly higher value (1.5 × 107) was obtained with a conidial formulation of Apopka-97. Average survival times were dosage dependent, i.e. between 10.2 days at 103 blastospores/ml and 3.5 days at 108 blastospores/ml. Rates of mycosis were also dosage dependent, with up to 100% sporulation on cadavers at 108 blastospores/ml but declining at lower concentrations. The Apopka-97 strain (commercially available as PFR-97) was tested against established D. citri infestations in potted citrus in greenhouse cages. Treatments at label rates reduced psyllid populations by approximately 50% over 3 weeks. The combination of PFR-97 with emulsifiable oils (0.25% v/v) did not increase psyllid mortality compared with either agent alone. Imidacloprid applied as a drench killed 100% of psyllids within 3 weeks. Subsequent greenhouse tests during humid conditions were hampered by natural dissemination of I. fumosorosea to untreated psyllids, suggesting that this fungus is spread by air movement and may be highly effective under very humid conditions. In later tests, a Cladosporium sp. rapidly colonised psyllid cadavers and leaf surfaces, but was not pathogenic in laboratory tests. Our studies confirm the potential of I. fumosorosea to be used in IPM strategies for D. citri that rely on other tactics, such as insecticidal oils and native or introduced biological control agents.

Compatibility of Isaria fumosorosea (Hypocreales: Cordycipitaceae) Blastospores with Agricultural Chemicals Used for Management of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae)

Biorational insecticides are being increasingly emphasized for inclusion in integrated pest management programs for invasive insects. The entomopathogenic fungus, Isaria fumosorosea, can be used to help manage the Asian citrus psyllid with minimal impact on beneficial arthropods, but its effectiveness may be compromised by agrochemicals used to control concurrent arthropod pests and diseases. We evaluated the compatibility of I. fumosorosea blastospores with a range of spray oils and copper-based fungicides registered for use in citrus groves. Results of laboratory and greenhouse tests showed a range of responses of the fungus to the different materials, including compatibility and incompatibility. Overall, I. fumosorosea growth in vitro was reduced least by petroleum-based materials and most by botanical oils and borax, and some of the copper-based fungicides, suggesting that tank mixing of I. fumosorosea with these latter products should be avoided. However, equivalent negative effects of test materials on fungal pathogenicity were not always observed in tests with adult psyllids. We hypothesize that some oils enhanced adherence of blastospores to the insect cuticle, overcoming negative impacts on germination. Our data show that care should be taken in selecting appropriate agrochemicals for tank-mixing with commercial formulations of entomopathogenic fungi for management of citrus pests. The prospects of using I. fumosorosea for managing the invasive Asian citrus psyllid and other citrus pests are discussed.

Effect of Isaria Fumosorosea (Hypocreales: Cordycipitaceae) and Lysiphlebus Testaceipes, (Hymenoptera: Braconidae) on the Brown Citrus Aphid: Preliminary Assessment of a Compatibility Study

Recently, a strain of Isaria fumosorosea Wize (.Ifr) (= Paecilomyces fumosoroseus [Luangsa-ard et al. 2005]), (Hypocreales: Cordycipitaceae) was discovered in a Florida citrus grove infecting the Asian citrus psyllid, Diaphorina citri Kuwayama (Meyer et al. 2008). Also, laboratory studies have demonstrated that Ifr may be effective against several commercially important pests of citrus (Poprawski et al. 1999; Avery et al. 2009; Hoy et al. 2010; Hunter et al. 2011). Therefore, the use of Ifr (PFR 97TM 20% WDG, Certis USA, Columbia, Maryland), approved in 2011 by EPA for use in commercial citrus, may offer a potential tool for balancing citrus pest management with consum er demands for organic citrus produce. The brown citrus aphid (BCA), Toxoptera citri cidus Kirkaldy (=T. citricida [Nieto Nafria et al. 2005]), (Hemiptera: Aphididae) was discovered in Florida in 1995 and is recognized as a serious pest of citrus capable of vectoring citrus triseza virus (Halbert & Brown 1996; Michaud 1998; Roy & Brlansky 2009). Under optimal conditions, na tive natural enemies provide good management of BCA, although these are easily disrupted by insecticide applications (Michaud 1998; 2002). Presently, the aphids population is managed in part by the parasitoid Lysiphlebus testaceipes Cresson (Hymenoptera: Braconidae) (Evans & Strange 1997). This generalist aphid parasitoid Lysiphlebus testaceipes, native to Florida and the Americas, is an effective biocontrol agent of BCA (Evans & Strange 1997). The objectives of our study were to assess the impact of Ifr on the mor tality of BCA populations as well as the parasit ism and emergence rates of L. testaceipes. BCA and Carrizo citrus (a trifoliate hybrid: Washington navel x trifoliate orange) were ob tained from the Sub-Tropical Insect Research Unit, USDA Horticulture Research Laboratory in Fort Pierce, Florida. For the experiment, 5 car rizo citrus seedlings (25-30 cm tall) were placed in each of four separate nylon mesh-covered cages (35 x 35 x 35 cm) and each infested with 4 alate BCA (20 per cage). A second infestation was made after 9 days. Cages were maintained in a growth room at 24.0 ± 1 °C, 45.0 ± 1 % RH and 16:8 h (L:D) photoperiod. Two weeks later, 10 L. testa ceipes adults were added to two of the cages. After an additional 4 days, one cage with and without L. testaceipes was treated with Isaria fumosoro sea (Ifr) suspension (PFR 97TM 20% WDG) at 107 CFU/ml using Nalgene® (Nalge Nunc Interna tional, Rochester, NY) aerosol sprayer. The ger mination of PFR 97TM blastospores was 96% using the technique of Avery et al. 2009. The remaining 2 cages were sprayed with an equal volume of wa ter. Parasitoids were temporarily removed from all cages prior to spraying to eliminate potential drowning and were replaced with an additional 5 to compensate for handling stress. Two replicates of the same study were conducted on different dates.

Evaluation of Entomopathogenic Fungi Against Chilli Thrips, Scirtothrips dorsalis

Abstract Commercial strains of entomopathogenic fungi were evaluated for control of chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae), an invasive pest of ornamental and vegetable plants in the Caribbean and southeastern United States. In laboratory assays, LC50 values against adult S. dorsalis were 5.1 × 104 CFU/mL for Beauveria bassiana GHA, with higher values 3.1 × 105 for Metarhizium brunneum F52 and 3.8 × 105 for Isaria fumosorosea Apopka 97. Second instars were comparatively less susceptible to all isolates, ostensibly due to moulting, with LC50 values of 1.1 × 108, 7.0 × 105, and 9.9 × 105 CFU/spores per mL for GHA, F52, and Apopka 97 strains, respectively. In greenhouse cages, compared with controls, three applications of mycoinsecticides and other biorational insecticides at 7 to 14 day intervals reduced overall S. dorsalis populations on pepper plants Capsicum annuum cv. California Wonder: spinosad reduced populations by 94–99%, M. brunneum F52 by 84–93%, B. bassiana GHA by 81–94%, I. fumosorosea PFR-97 by 62–66%, and different horticultural oils by 58–85%. The proportion of marketable fruit was significantly increased by M. brunneum F52, B. bassiana GHA, and 2% SuffOil-X treatments. Slightly lower levels of control were observed in nursery tests with ornamental rose shrubs, Rosa sp. Red Double Knock Out®, during hot sunny conditions. Four applications reduced thrips populations over 10 weeks: spinosad by an average of 91%, M. brunneum F52 by an average of 81%, B. bassiana GHA by an average of 62%, SuffOil-X by an average of 50%, and I. fumosorosea PFR-97 by an average of 44%. The data show that mycoinsecticides can be used in management strategies for low to moderate populations of S. dorsalis and provide resistance management tools for the limited number of insecticides that are effective against this pest.

Effect of Pollens of Various Ornamental Pepper Cultivars on the Development and Reproduction of Amblyseius swirskii (Acari: Phytoseiidae)

Abstract The rationale behind the current study was to assess if the commercially available generalist phytoseiid mite, Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), can survive and reproduce when fed on pollen from ornamental pepper (Capiscum annum L.; Solanales: Solanaceae). Results from this study will help in the selection of ornamental pepper cultivars as candidate banker plants for establishment of A. swirskii in ornamental nurseries. To determine the effect of different types of pepper pollen on survival and multiplication ability of mites; their longevity, daily rate of oviposition and total egg deposition was assessed. Amblyseius swirskii was reared on pollen collected from 4 ornamental pepper cultivars namely ‘Red Missile’, ‘Masquerade’, ‘Black Pearl’ and ‘Explosive Ember’. Amblyseius swirskii was able to survive, develop and oviposit when fed with pollens of all the 4 ornamental pepper cultivars and the 2 standard controls i.e., commercially available olive pollen Olea europaea L. (Lamiales: Oleaceae) and Scirtothrips dorsalis Hood (Thysanoptera: Thripidae). No significant differences were observed among the various diet treatments for the following biological parameters: mean duration from larvae to adult death (22.8–23.7 days), duration from larvae to eggs (11.8–12.5 days) and adult longevity (12.4–13.1 days). However, for mites fed on pollen the highest daily rate of oviposition (∼1.07 eggs/female/day) and total oviposition (∼10.23 eggs/female) was recorded on the ‘Red Missile’. Outcome of this study can boost the management strategies being used against S. dorsalis and other pest species affecting ornamental plants in Florida.

Volatiles from Plants Induced by Multiple Aphid Attacks Promote Conidial Performance of Lecanicillium lecanii.

Herbivore-induced plant volatiles (HIPVs) are clues that help predatory insects search for food. The hypothesis that entomopathogenic fungi, which protect plants, benefit from the release of HIPVs was tested. The plant Arabidopsis thaliana was used as the source of HIPVs. The insect herbivore Lipaphis erysimi (Kaltenbach) was used as the inducer, and the fungal pathogen of the aphid Lecanicillium lecanii was exposed to HIPVs to test our hypothesis. When exposed to aphid-induced A. thaliana volatiles, the mortality of aphids pre-treated with a conidial suspension of L. lecanii, the conidial germination and the appressorial formation were significantly increased compared with the control. The decan-3-ol and 4-methylpentyl isothiocyanate that were detected in the headspace seemed to have positive and negative affection, respectively. Moreover, HIPVs generated from groups of eight aphids per plant promoted significantly increased conidial germination and appressorial formation compared with HIPVs from groups of one, two and four aphids per plant. Our results demonstrated that the pathogenicity of the entomopathogenic fungus L. lecanii was enhanced when exposed to HIPVs and that the HIPVs were affected by the number of insect herbivores that induced them.

Compatibility and Efficacy of Isaria fumosorosea with Horticultural Oils for Mitigation of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae)

Horticultural oils are an important component of integrated management programs of several phytophagous arthropods and pathogens affecting fruit, ornamentals and vegetables in greenhouse and field production systems. Although effective against the target pest, their incompatibility with biological control agents can compromise efforts to develop eco-friendly management programs for important agricultural pests. In this study, we assessed the in vitro effect of selected refined petroleum oils used in citrus and other horticultural crops with a biopesticide containing the entomopathogenic fungi, Isaria fumosorosea (PFR-97) under laboratory conditions. Further, we used leaf disk bioassays to evaluate the combined efficacy of petroleum oils and I. fumosorosea against the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), a major pest of citrus in the United States. All five petroleum oil treatments (Orchex, Sun Pure, Conoco Blend -1, Conoco Blend -2, and JMS) were compatible with I. fumosorosea blastospores, as none of them were found to affect I. fumosorosea colony-forming units and radial fungal growth measured at 3, 6, 9, and 12 days post-inoculation. All mixed treatments performed better than I. fumosorosea alone against D. citri, where the highest mean survival time of D. citri was 12.5 ± 0.7 days. No significant differences in D. citri survival time and I. fumosorosea growth (fungal development index) on dead cadavers, which is important for determining their horizontal transmission, were observed when mixed with Orchex, Sun Pure, Conoco Blend -2, and JMS. Results indicated that horticultural oils in combination with I. fumosorosea could offer citrus growers an alternative treatment for integrating into their current management programs while battling against D. citri in citrus production systems. Due to their eco-friendly, broad-spectrum effect, it could provide control against various citrus pests, while also encouraging the retention of effective chemistries for a longer period in the marketplace. However promising, these combination treatments need to be tested further with I. fumosorosea under grove conditions to confirm their field efficacy.