Renewed interest in the use of entomopathogenic 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 invasive, Asian citrus psyllid, Diaphorina citri (Kuwayama) (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 orange 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 laboratory conditions. The Asian citrus psyllid, D. citri, is the vector of the citrus-infecting bacteria associated with citrus yellow shoot disease, known as Huanglongbing (Bové, 2006; 2008), and the glassy-winged sharpshooter, Homalodisca vitripennis Germar (Hemiptera: Cicadellidae) is a vector for the plant-infecting bacteria, Xylella fastidiosa which causes citrus variegated chlorosis, scorch-like diseases of fruit crops, and Pierce's disease of grapevine (Daugherty & Almeida 2009). the brown citrus aphid, Toxoptera citricidus (Kirkaldy) (Hemiptera: Aphididae), is a vector 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 entomopathogenic fungus, Ifr.

Presently, 3 Ifr strains are available for research as blastospore formulations in the U.S.A. There is the Ifr APOPKA strain, in PFR 97 20% WDG® (Certis, Columbia, Maryland, USA), labeled for controlling psyllids, leafhoppers, and aphids; the Ifr 9901 strain, in NoFly™ WP (Natural Industries, Houston, Texas labeled for controlling 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 characteristics 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 conspecifics (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 conditions.

Effects on insects were assessed under laboratory conditions. Plants used in all these experiments were either Duncan grapefruit (Citrus paradisi Macf.) or ‘carrizo’ citrus seedlings grown in Premier Pro-mix General Purpose Growing Medium in size C10 “Cone-tainers”™ (Stuewe & Sons, Inc., Corvalis, Oregon) from seed for approximately 6 months. Experiments that used leaf disks, leaves or seedlings were sprayed at the same rate (10⁷blastospores / ml) for all insects. Insects were all kept at 25 °C under a 16:8 h L: h D photoperiod. Diaphorina citri and Homalodisca vitripennis: Leaf disks (∼962 mm²) cut to fit snugly in a 35 × 10 mm Petri dish, were sprayed with Ifr (PFR 97) to runoff (1,313 ± 257.7 blastospores mm²) using a Nalgene® aerosol sprayer (Nalge Nunc International, Rochester, New York) or water and then allowed to air dry in a fume hood. Disks were then placed on top of warm water agar and allowed to stick to the agar after the leaf was embedded, then 1 adult psyllid was allowed to walk off the camel hair brush onto the leaf disk in each dish. Sixty dishes (control and treated) were sealed with Parafilm™ for 24 h and then left unsealed thereafter, but held together by a rubber band (RH was not measured inside the dish) (Lietze et al. 2011). Control leaf discs were treated similarly using only water, and insects were added as with treatments with corresponding numbers of insects. Insects were monitored daily until death occurred and mycosis of Ifr was verified by characteristic morphology of the fungus inside each dish (Fig. 1 A–B). Glassy winged sharpshooter nymphs (2 – 3) or adults (2) per dish were monitored as described above (Fig. 1 C–D). Leafhopper egg masses were also treated and observed (Fig. 1 E–F). Toxoptera citricidus: Two bug dorm cages (35 cm³), were set up with 2 cages per light fixture (four 40W Philips fluorescent lights) under a 16 h L: 8 h D photoperiod at 24 ± 0.03 °C with 45 ± 0.29% RH. Five carrizo citrus seedlings (25–30 cm tall) grown in 4.9 cm³ yellow Cone-tainers® held in a rack were added to each cage, and watered as needed. Citrus seedlings were sprayed with Ifr or water, allowed to air dry, and then 20 adult aphids were added to roam freely (Fig. 1G). Dead aphids were removed daily from cages, and/ or leaves, and placed on water agar plates to determine if they were infected with Ifr, verified by characteristic morphology of the fungus (Fig. 1H) (Poprawski et al. 1999; Pick et al. 2010).

Figure 1.

Citrus insect pests exhibiting mycosis of Isaria fumosorosea: A) Fungi growing on leaf surface, scanning electron micrograph; B) Asian citrus psyllid showing extrusion of fungal hyphae; C) Adult glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae); D) Leafhopper showing extrusion of fungal hyphae. Nymphs were also susceptible to fungal spray treatments (not shown). Leafhoppers died while feeding on citrus seedling 6–7 d post treatment sprays of PFR 97™. E) Healthy leafhopper egg mass covered in whitish powder of brocosomes. F) Treated leafhopper egg mass in citrus leaf showed fungi hyphae at oviposition sites (arrows); nymphs did not emerge. G). Citrus aphid, Toxoptera citricidus, apterous adult when given access to citrus leaf treated with PFR 97™, H) become comatose, slowly stops feeding and are colonized by fungi. I) Diaprepes citrus root weevil, showing signs of Ifr colonization at day six post treatment. J) Fully mycosed weevil adult.

Diaprepes abbreviatus adult weevils were fed single grapefruit leaves of similar size that were sprayed with either Ifr or water, and allowed to air dry in a fume hood. One adult was placed in a 100 × 15 mm Petri dish containing a single leaf; 40 adults per treatment. High humidity (∼100% RH) was provided by a moistened filter paper on the bottom of the dish. Dish chambers were sealed with Parafilm™ and placed in a growth chamber for several weeks at 25 °C under a 16 h photophase. Once a leaf was consumed after 24 h, an untreated leaf was provided to the adult weevils and replaced thereafter as needed until death occurred. Adults were monitored daily for 1–6 weeks for symptoms of the fungus (Fig. 1 I–J).

The percentage mortality for the treated insects is summarized in Table 1. Psyllids appeared most susceptible to PFR 97™ with the earliest mortality observed 2 d post-exposure, with 100% mortality by the eighth d. During this time period, psyllid feeding was reduced as evidenced by the reduction of honeydew droplets (Avery et al. 2011). Leafhopper adults showed increasing mortalities beginning 3–4 d up to 100% at 15 d post exposure. The glassy-winged sharpshooter leafhopper is significantly larger than the psyllid (∼4 *), which may account for the delayed mortality rates, plus leafhoppers continuously groom with their spiky hind legs which could dislodge spores from penetrating the cuticle. Leafhopper nymphs showed similar rates of mortalities during the same time period, with 100% still occurring at 15 d post exposure. Aphids showed 100% mortality at 15–20 d post exposure. Within all hemipteran control groups, mortality was 3% or less over the same time period. For the coleopterans, weevils have a much larger body size, with a thicker cuticular layer that could potentially increase the difficulty for fungi to penetrate. In weevil control groups, 13% mortality was observed 15 d post-exposure reaching a maximum of 33% at 35 d, while fungal treated weevils showed 100% mortality at 35 d post exposure.

TABLE 1.

PERCENT MORTALITY (RANGE) AND TIME OF DEATH OF VARIOUS INSECTS AFTER BEING EXPOSED TO ISARIA FUMOSOROSEA.

The Ifr spray product, i.e. PFR 97™, demonstrated that applications of these strains have potential to reduce citrus insect pests and may provide an additional biological control agent; however, these results need to be confirmed under field conditions. Furthermore, the role of environmental conditions, such as early morning humidity, rainy seasons, or temperatures are being investigated for their impact on efficacy (Avery - unpublished observations) to establish best management practices and integration with currently implemented IPM practices in citrus production.

SUMMARY

The insects evaluated were susceptible to infection by the Hypocreales fungus, Ifr, in the product PFR 97™ and Ifr 3581 when applied as a spray to seedlings, leaves or leaf discs. This is the first report indicating that H. vitripennis, T. citricidus and D. abbreviatus were susceptible to Ifr strains under laboratory conditions. The results suggest that Ifr provides a potential biological control agent for these and other pests of agricultural crops, and may provide a product for organic growers, as well as an additional tool to augment standard IPM programs.