Plant culture

Oryza sativa L. (Poales: Poaceae) cv. Nongfengyou, a rice cultivar susceptible to N. lugens, was prepared: 10-day-old rice seedlings in dishes (15 cm diameter) were transplanted in plastic pots (20 cm diameter, 20 cm high), which were filled with nutrition solution and cultured for another 10 days under controlled conditions (25–30°C; 14:10 L:D photoperiod; 80% RH) in the greenhouse at the School of Plant Protection, Anhui Agricultural University, Hefei, China.

N. lugens population rearing

Adult N. lugens (Figure 1A), originally collected from the rice fields in Anhui Agricultural University (No. 130 West Changjiang Rd, Hefei, Anhui, China), were reared on the rice seedlings described above. The condition stimulated the copula and oviposition by females. Nymphs that were born from these ovipositions and grew up to be adults were used in pathogenicity assays.

Sources of fungi

The fungal isolates of B. bassiana and B. brongniartii (Saccardo) used in the experiment were provided by Biocontrol Reserch Lab, Anhui Agricultural University, Hefei, China, and originally isolated from a broad spectrum of insects (Table 1). In order to confirm virulence of the isolates was not attenuated, original host insects were inoculated with these isolates and the fungi were reisolated on Sabouraud dextrose agar yeast medium. The conidia colonies were inoculated to Sabouraud dextrose agar yeast medium in Petri dishes (9 cm diameter) and maintained at 25 ± 1°C in darkness for 15 days. After being scraped from the plate, conidia were dried to a water content of 5% at ambient temperature with a vacuum drier (VirTis, SP Scientific,  www.spscientific.com). Dry conidia were preserved at 4°C in darkness for use as soon as possible in all tests, warranting ≥ 90% viability.

Conidia of each fungal isolate were transferred into corresponding test tubes (3 cm diameter, 20 cm length) and suspended in sterile distilled water by shaking in 10 mL flasks containing 60 to 72 glass beads (3 mm diameter). No surfactant [0.02% Tween-80 (polyoxyethylene sorbitan monolaurate)] was added, since 5 min of agitation at 700 oscillations on a mechanical shaker homogenize suspensions of viable single conidia. The suspensions were then adjusted to 1 × 10⁸ conidia/mL by appropriate dilution based on hemocytometer counts.

Bioassays with N. lugens adults

Two different series of bioassays were conducted. For each fungal isolate, 60 adults (approximately 10 days old) from the rice seedlings in the greenhouse were aspirated into test tubes (3 cm diameter, 20 cm length). Afterwards, they were transferred into a transparent bell glass jar (6 cm diameter, 20 cm high) with a bottle cap that had small holes for air circulation to avoid adults escaping. The suspensions of 1 × 10⁸ conidia/mL in sterile water with 0.05% (v/v) Tween-80 that was prepared previously were sprayed onto adults and rice seedlings in the glass jar by a hand-held Micro Ultra sprayer (Micron Sprayers,  www.micron.co.uk). The concentration of conidia deposited onto the adults and seedlings was measured as number of conidia/mm² using microscopic (400 × magnification) counts of conidia collected onto four glass slips (20 × 20 mm) under each spray. Four replicates per isolate were used, and a blank control was sprayed with 0.05% (v/v) Tween-80 for each fungal isolate. All treatments were maintained at 25 ± 1°C, a 14:10 L:D photoperiod, and 80% RH, with adult mortality recorded daily for 10 days post-treatment. The dead adults were transferred every day into 70% ethanol for 10 sec, washed in sterile distilled water, then treated with 0.5% sodium hypochlorite for 30 sec, and washed again in sterile distilled water. After that, the specimens were placed in Petri dishes (9 cm diameter) with filter paper that was moistened with sterile distilled water and maintained in a humidity chamber (saturated atmosphere) for fungal sporulation at 25 ± 1°C for three to five days.

The second series of bioassays included only those isolates that caused greater than 50% adult mortality in the first series of experiment. Bioassays used aqueous B. brongniartii (Bbr03 and Bbr09) suspensions (1 × 10⁶, 1 × 10⁷ , and 1 × 10⁸ conidia/mL) containing 0.02% Tween-80 in distilled water (the control). The bioassays were repeated four times using the same method described above.

Fungal characteristics, colony growth rates, and conidial yields

The isolates were cultured on Sabouraud dextrose agar yeast medium in Petri dishes (90 mm diameter), with four replicates for each isolate. The dishes were maintained in an incubation chamber at 25 ± 1°C and RH ≥ 80% in the dark. The diameters of the colonies were measured five, 10, and 15 days after incubation.

After 15 days, a culture disc from the center of each fungal colony was removed using a sterile 13 mm diameter punch and placed into a 50 mL flask with 20 mL sterile 0.05% Tween-80. Each isolate contained four flask cultures as replicates. The conidial suspensions of the nine isolates were prepared by stirring as described above. Conidial concentrations were determined using a hemocytometer according to the method by Zhang et al. (2011).

Conidial germination

Fifteen µL of each conidial suspension were transferred to a sterile glass slide smeared with Sabouraud dextrose agar yeast. The slide was incubated in at 25 ± 1°C and RH > 90%. Four replicates were set up for each isolate. Conidial germination was estimated at 24 hr by counting the germinated and non-germinated conidia in a random field of vision sample on each slide using a microscope at 400× magnification. The number of germinated and non-germinated conidia from four microscopic view fields (100 conidia/field) was counted. For each observation, 400 conidia were examined in each replicate slide. The mean percent germination was calculated for each sample.

Chitinase activity assay

The enzymatic reaction was initiated by addition of 200 µL isolated chitinase solution to the colloidal chitin (10% w/v chitin in 300 µL acetate buffer, pH 5.0) as substrate and continued for 60 min at 50°C. The hydrolysis of chitin was measured by the para-dimethyl-amino benzaldehyde reagent method according to Reissig et al. (1955).

The reaction was cooled to room temperature, centrifuged at 5,000 × g for 10 min, and the absorbance at 540 nm (A₅₄₀) was taken against water as blank. The enzyme activity was measured according to a standard curve developed using known concentrations of N-acetyl-d-glucosamine. One enzyme unit (U) was defined as the amount of enzyme that produced 1 µM of N-acetyl glucosamine per minute under the above conditions.

Table 2.

Bioassay results for adults of Nilaparvata lugens representing isolates of Beauveria.

Statistical analyses

The mortalities (%) of N. lugens in treatments (M2) were corrected by the mortality (%) in blank controls (M1) according to Abbortt's formula: Mc = (M2 - Ml) / (100-M1). All corrected mortalities (%) data were subjected to an arc-sine transformation prior to analysis of variance to detected differences in mortalities of N. lugens. Mean separation was performed using Tukey's honestly significant difference test (p <0.05). Statistical analyses were performed using the DPS program (Tang and Feng 2007).

Infection of fungal pathogens to N. lugens adults

It was found that N. lugens adults infected with Beauveria moved sluggishly in the early stage of infection, female adults stopped oviposition, and nymphs had difficulty in peeling. The infected N. lugens adults then died holding rice stems, which were not found in the control group (Figure 1A). As the disease progressed, N. lugens adults' bodies turned brown when infected with Beauveria for three days, and white flocculent hyphae grew out of the insect body from the segmacoria, somatic fold sag, foot joints, etc. In the following one to two days, the mycelium covered the whole body until white or light yellow conidia (Figure 1B) emerged. From inoculation to the completion of the entire process of intrusion infection, tissue lesions, host's death, and in vitro sporulation, the shortest time needed was four days, and the longest nine days.

Table 3.

Virulence of the selected isolates Bbr03 and Bbr09 towards Nilaparvata lugens adults.

Table 4.

Characteristics of the tested Beauveria bassiana (Bb) and B. brongniartii (Bbr) isolates on medium.

Virulence test on N. lugens adults

The cumulative corrective mortalities of N. lugens differed significantly among the 10 isolate treatments (F_{8, 27} = 269.5, p < 0.01) on the 10th day after spraying. Only two isolates, Bbr03 and Bbr09, killed more than 50% of the adults, of which Bbr09 had the lowest LT₅₀ of (5.5 days). Cumulative adult mortality increased with conidial concentration. Based on the estimates of the LC₅₀ and associated 95% CI (Table 3), the two isolates, Bbr09 and Bbr03, had LC₅₀S of 134 and 443 conidia/mm² on day 10, respectively.

Fungal characteristics, colony growth rates, and conidial yields

Viewing from biological characteristics of the original 15 days of culture (Table 4), colony growth rates (5 days: F_{8, 27} = 60.7, p < 0.01; 10 days: F_{8, 27} = 177.1, p < 0.01; 15 days: F_{8, 27} = 247.4, p < 0.01), conidial yields (F_{8, 27} = 477.7, p < 0.01), and spore germination (F_{8, 27} = 31.8, p < 0.01) of the nine isolates were tested, and the results differed significantly among them. The isolate Bbr09 generally had the fastest colony growth rates and the highest conidial yields. Spore germination of all tested isolates exceeded 80% within 24 hr.

Figure 2.

Chitinase activity assay of the examined strains. The letters on the top of columns show the significance of difference. High quality figures are available online.

Chitinase activities

The chitinase activities of nine isolates demonstrated a significant variation (Figure 2; p < 0.01). The three strains Bbr09, Bbr03, and Bb255 had high chitinase activities, among which Bbr09 had the highest chitinase activity of (18.7 U/mL), whereas, the isolate Bb81 showed the least chitinase activity (4.3 U/mL).