In vivo passages of heterologous Beauveria bassiana isolates improve conidial surface properties and pathogenicity to Nilaparvata lugens (Homoptera: Delphacidae)

Journal of Invertebrate Pathology 106 (2011) 211–216

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In vivo passages of heterologous Beauveria bassiana isolates improve conidial surface properties and pathogenicity to Nilaparvata lugens (Homoptera: Delphacidae) Ting-Ting Song, Ming-Guang Feng ⇑ Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 7 July 2010 Accepted 28 September 2010 Available online 27 October 2010 Keywords: Nilaparvata lugens Beauveria bassiana Heterologous host passage Conidial hydrophobicity Zeta potential Cuticle-degrading proteases Fungal pathogenicity

a b s t r a c t In entomopathogenic hyphomycetes, desired candidates against the brown planthopper, Nilaparvata lugens (a sap-sucking rice pest in Asia), are lacking. In this study, 21 Beauveria bassiana isolates from heterologous host insects showed low pathogenicity to third-instar nymphs sprayed at the high concentration of 1000 conidia/mm2, causing only 2–23% mortalities. Of those, three isolates killed significantly more nymphs (up to 45–62%) after two in vivo passages but no more after further passage. Conidial hydrophobicity rates (Hr), zeta potentials (Pz), and subtilisin-like protease (Pr1) activities (Ap) of these isolates showed the same trends in the three host passages (N: 0–3). In multivariate correlation, the variables N, Hr and Pz were found contributing 89% to the mortality variation (r2 = 0.89). Significant positive correlations were also found between Hr and N (r2 = 0.64), Pz and N (r2 = 0.52), Ap and N (r2 = 0.51), Hr and Ap (r2 = 0.45), and Pz and Ap (r2 = 0.57), respectively. However, irregular changes of Hr and Pz occurred in four other isolates, whose pathogenicity to N. lugens was not enhanced by repeated host passages, resulting in no correlation between the variables. Our data indicate that the conidial surface properties Hr and Pz associated with cuticle adhesion reflect the heterologous host-induced adaptation and help to select fungal candidates against N. lugens from repeated in vivo passages. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction The brown planthopper, Nilaparvata lugens Stål, is a major sapsucking rice pest in Asia (Backus et al., 2005) and has developed high resistance to a variety of chemical insecticides including neonicotinoid compounds, such as imidacloprid (Liu et al., 2005; Liu and Han, 2006). Transgenic rice lines expressing Bacillus thuringiensis crystal endotoxins that kill lepidoperan stem borers or leaf rollers have little effect on N. lugens (Bernal et al., 2002). An alternative strategy for the pest control is the use of fungal biocontrol agents, such as Beauveria bassiana and Metarhizium anisopliae (Feng and Pu, 2005; Jin et al., 2008). In a large number of isolates assayed in early 1980s, unfortunately, very few were found to kill >70% rice planthoppers (Roberts and St. Leger, 2004). Recently, 35 isolates of Metarhizium spp. with different host and geographic origins were bioassayed under the spray of a concentrated spore suspension and only two of them (both M. anisopliae) were able to cause 60% mortality of N. lugens nymphs (Jin et al., 2008). Similarly, a single isolate was found to kill 50–73% in the bioassays of 17 B. bassiana isolates on three species of planthoppers and leafhoppers (Toledo et al., 2007). These data contrast with the ease of attaining ⇑ Corresponding author. Fax: +86 571 8820 6178. E-mail address: [email protected] (M.-G. Feng). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.09.022

fungal candidates with high pathogenicity to other sap-sucking pests, such as aphids, whiteflies and spider mites, through routine bioassays (Vandenberg, 1996; Wraight et al., 1998; Shi et al., 2008). Fungal pathogenicity to a target insect pest varies largely among species and isolates (Feng et al., 1994; Roberts and St. Leger, 2004) and can be enhanced by passing isolates through host species (Fargues and Robert, 1983). Serial in vitro transfers of fungal isolates can alter phenotypic features and may, but not necessarily, affect their pathogenicity to an original or heterologous host (Brownbridge et al., 2001; Vandenberg and Cantone, 2004). In one study, no change in RAPD banding pattern using 14 sets of primers in PCR, was observed despite changes in pathogenicity and/or phenotypic features of host specificity, growth, and conidiation after 30 passages of the strains examined (Vandenberg and Cantone, 2004). Nevertheless, fungal infection always starts from conidial adhesion to host cuticle, followed by germination and cuticle penetration into haemocoel (Feng et al., 1994; Sosa-Gomez et al., 1997). Cuticle adhesion is affected by both conidial hydrophobicity and electrostatic status (Boucias et al., 1988; Holder and Keyhani, 2005; Cho et al., 2007). Hydrophobicity is often assessed with the method of aqueous-solvent partitioning (Holder et al., 2007; Shah et al., 2007; Shan et al., 2010). Electrostatic status can be measured as the zeta potential of conidial surface, which is the overall charge at the outermost layer of electrical double layer

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Twenty isolates of B. bassiana (denoted Bb in this report) with different host and geographic origins (Table 1) were from the ARS Collection of Entomopathogenic Fungal Cultures (ARSEF; RW Holley Center for Agriculture and Health, Ithaca, NY, USA). These ARSEF isolates, together with one local isolate (Bb0201), were preserved at 76 °C and recovered on the plates of Sabouraud dextrose agar plus 1% yeast extract (SDAY) for 7 days at 25 °C prior to use, followed by routine maintenance at 4 °C during the study. For each of the isolates, aerial conidia were produced by incubating on SDAY at 25 °C and 12:12 L:D for 7–9 days, harvested by scraping, and suspended in 0.02% Tween 80 at the standardized concentration of 1  108 conidia/ml for bioassays.

standard method described elsewhere (Feng and Pu, 2005; Jin et al., 2008). Thirty to 40 nymphs taken from the tray were transferred onto 30 3-cm-tall seedlings individually growing upwards from the pores of a sponge board floating in a plastic cup (7 cm diameter, 9 cm height), in which nutrition solution was filled for rice root growth. The conidial suspension of each isolate (prepared on the same day) was sprayed onto the nymphs and seedlings for 30 s using a hand-held Micro Ulva Sprayer (Micron Sprayers Limited, Herefordshire, UK), which generated mist droplets of 50– 60 lm at labeled working pressure. After 5 min deposition, the seedlings were gently covered with a top-meshed cage and maintained for 10 days in growth chamber at 25 °C and 12:12 L:D. The nymphs on the seedlings were monitored daily for mortality and cadavers were transferred into saturated petri dishes for fungal outgrowth and sporulation. The concentration of the conidia deposited onto the nymphs and rice seedlings under each fungal spray was determined as the number of conidia per square millimeter using five microscopic counts from a glass coverslip (20  20 mm) placed beside each cup of seedlings for spore collection. The bioassay was repeated three times and the same spray of 0.02% Tween 80 only was used as blank control. Single-spore isolates were prepared from N. lugens cadavers killed in the initial bioassay and assayed on the third-instar nymphs under the same conditions using the methods of conidial production and suspension preparation as described above. The in vivo passage and bioassay of the representative isolates selected from previous bioassay were repeated until they caused no more significant mortality increase under the same concentrated spray. After the initial bioassay, three in vivo passages and bioassays were consecutively performed, including 11, 7 and 3 isolates, respectively. The isolates derived from the second or third in vivo passage and their counterparts used in the previous bioassays were chosen for assessing their conidial surface properties and Pr1 activities as follows. Aerial conidia of these isolates were produced on SDAY plates, vacuum dried to a water content of 5% at ambient temperature after harvest, and then stored in sealed glass vials at 4 °C for use.

2.2. Bioassays

2.3. Measuring conidial hydrophobicity

All 21 isolates were initially bioassayed on the third-instar nymphs of N. lugens reared in a tray of rice seedlings with a

Conidial hydrophobicity rates (Hr) of the selected isolates were assessed using the modified method of aqueous-solvent

surrounding all surfaces of conidia within an aqueous environment (Giradin et al., 1999). After conidial adhesion and germination, cuticle penetration relies mainly upon a variety of cuticle degrading enzymes (Clarkson and Charnley, 1996). Of those, subtilisinlike proteases (Pr1) are cuticle-degrading virulence factors (Wang et al., 2002; Freimoser et al., 2003; Dias et al., 2008), which are associated with conidial surface properties (Shah et al., 2007). However, none of these physiochemical surface properties has been related to changes in fungal pathogenicity after in vivo passages. To search potential candidates against N. lugens, we evaluated 21 heterologous B. bassiana isolates for their pathogenicity to the target pest. All tested isolates initially caused low mortalities. However, three isolates displayed progressively increased virulence as the heterologous host passage proceeded. Thus, we correlated alternations in conidial hydrophobicity, zeta potential and Pr1 activity to sequential in vivo passages through N. lugens. Our data would help to understand the importance of the altered surface properties for the increase of fungal pathogenicity by heterologous host-induced adaptation. 2. Materials and methods 2.1. Fungal isolates and conidial preparations

Table 1 Host and geographical origins of Beauveria bassiana (Bb) isolates for bioassays.

a

Isolatea

Original host insect

Geographic origin

Bb0201 Bb156 Bb734 Bb1269 Bb1395 Bb2860 Bb2861 Bb2864 Bb2879 Bb2880 Bb2881 Bb2882 Bb2883 Bb2988 Bb3070 Bb3312 Bb3620 Bb4535 Bb4580 Bb5705 Bb5965

Empoasca vitis [Homoptera: Cicadellidae] Species unknown [Hymenoptera: Ichneumonidae] Chalcodermus aeneus [Coleoptera: Curculionidae] Leptocoris oratorius [Hemiptera: Rhopalidae] Lygus sp. [Hemiptera: Miridae] Schizaphis graminum [Homoptera: Aphididae] Diuraphis noxia Homoptera: Aphididae] Diuraphis noxia[Homoptera: Aphididae] Diuraphis noxia [Homoptera: Aphididae] Schizaphis graminum [Homoptera: Aphididae] Schizaphis graminum [Homoptera: Aphididae] Schizaphis graminum [Homoptera: Aphididae] Schizaphis graminum [Homoptera: Aphididae] Leptinotarsa decemlineata [Coleoptera: Chrysomelidae] Aphodius tasmaniae [Coleoptera: Scarabaeidae] Species unknown [Homoptera: Membracidae] Melanoplus sp. [Orthoptera: Acrididae] Plutella xylostella [Lepidoptera: Plutellidae] Species unknown [Orthoptera: Acrididae] Diuraphis noxia [Homoptera: Aphididae] Solenopsis saevissima [Hymenoptera: Formicidae]

Menghai Co., Yunnan, China Poland Goiânia, Goiás, Brazil Palawan, Philippines Seine-Saint-Denis, France Parma, Idaho, USA Parma, Idaho, USA Parma, Idaho, USA Parma, Idaho, USA Parma, Idaho, USA Parma, Idaho, USA Parma, Idaho, USA Parma, Idaho, USA Sainte-Clotilde, Québec, Canada South Island, New Zealand Cuauhtémoc, Colima, Mexico USA Ontario Co., New York, USA Tasmania, Australia Western Cape, South Africa Lavras, Minas Gerais, Brazil

All isolates were coded with ARSEF accession numbers except the Chinese isolate Bb0201.

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partitioning (Shan et al., 2010). Briefly, 40 ll of liquid paraffin, often used as an oil vector of fungal conidia for formulation (Feng and Pu, 2005), was added to 4 ml of conidial suspension (2  106 conidia/ml) in the buffer of 1 M KH2PO4–K2HPO4 (pH 7.14). The mixture in 20-ml standard separation funnel was vortexed for 3 min, followed by a short wait for separation of the organic and aqueous phases. Three aliquots were then pipetted into haemocytometers from the aqueous phase. The residual conidial concentration (C) in the aqueous phase was determined using microscopic counts. For each isolate, Hr was finally assessed as (1 C/C0)  100, where C0 was the conidial concentration in the initial aqueous suspension not containing paraffin. 2.4. Measuring conidial zeta potential Conidia of each selected isolate were suspended in distilled water at the concentration of 1  107 conidia/ml. The zeta potential of conidia in the suspension (500 ll in 1 ml sample cup) was measured as electrophoretic mobility under the 10 V electric field of JS94H electro-kinetic analyzer (Zhongchen Digital Technical Apparatus Co., Shanghai, China). The electrophoretic mobility was estimated by the analysis of video-captured images using the JS94H software. The conidia suspending in distilled water (pH 7.0) were well distributed under the vibrating action of the electric field, thereby excluding possible pH and ionic effects on the surface charge but not interfering with the measurement. Each of three conidial samples per isolate was repeatedly assayed 15 times (i.e., measuring from 15 images).

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3. Results 3.1. BPH mortalities caused by B. bassiana Under the standardized sprays, the concentrations of the conidia deposited onto N. lugens nymphs on rice seedlings were averaged as 1065 ± 218 conidia/mm2 (n = 21) in the initial bioassay, and 1043 ± 231 (n = 11), 1063 ± 106 (n = 7) and 1053 ± 84 (n = 3) conidia/mm2 in the bioassays after the first, second and third in vivo passages, respectively. These estimates did not differ significantly from one bioassay to another (F3,38 = 0.68, P = 0.57). Corrected mortalities caused by the tested isolates in the first three bioassays are shown in Fig. 1. In the initial bioassay, all 21 heterologous isolates displayed low pathogenicity to N. lugens, causing mortalities from 2.4% (Bb5965) to 23.1% (Bb2881). The mortalities differed significantly among the 21 isolates (F20,42 = 2.1, P = 0.021). Under the same concentrated spray, some of the isolates derived from the first passage killed significantly more nymphs (up to 39.3%) than others (F10,22 = 2.3, P = 0.047), which caused similar mortalities as observed previously. Three isolates (i.e., Bb2860, Bb2861, and Bb2879) from the second passage became significantly more pathogenic to the pest species in the third bioassay (F6,14 = 13.8, P < 0.0001), causing 45–62% mortalities, but did not kill more nymphs after the third passage (Table 2; Fisher’s LSD, P > 0.05). However, significant mortality changes were not found among the three bioassays of four other isolates (Bb734, Bb1269, Bb2864, and Bb4535) irrespective of passing them through N. lugens once or twice or not (Table 3; P > 0.05 in oneway ANOVA).

2.5. Assaying conidial Pr1 activity For each of the selected isolates, conidial Pr1 activity was assayed using a method described by Shah et al. (2005). Briefly, 10 mg aliquots of dry conidia were washed once in 0.3% Tween 80 and twice in distilled water, followed by centrifugation at 8000g. The deposits were resuspended in 1 ml of 0.1 M Tris– HCl (pH 7.95) containing 1 mM succinyl-ala-ala-pro-phe-p-nitroanilide (Sigma) as substrate and incubated for 5 min at ambient temperature. The suspension was then centrifuged at 12,000g for 5 min. The supernatant was transferred to three wells (200 ll each) of a microtitre plate and spectrophotometrically assayed for the Pr1 activity at ambient temperature by reading OD405. The Pr1 activity in each conidial sample was defined as the amount of Pr1-reduced substrate per milliliter per minute (lM ml 1 min 1). Each of three samples per isolate was assayed three times. The substrate inclusive buffer was included as blank control.

3.2. Effects of in vivo passages on conidial surface properties and Pr1 activities Listed in Table 2 are N. lugens mortalities, conidial Pr1 activities, hydrophobicity rates, and zeta potentials of the three isolates for separate comparisons across the three in vivo passages. For each isolate, all the examined parameters were significantly enhanced by each sequential host passage compared to the initial low levels (mostly P < 0.001, occasionally P < 0.05 in the F tests of one-way ANOVA across the in vivo passages). Note that the differences between the second and third passages were not significant for most of the means (Fisher’s LSD, P > 0.05). This suggests that each variable become relative stable after the second passage. For other four isolates passaged twice through N. lugens, only Pr1 activity significantly increased after one or two passages (Table 3). Such a trend was not observed in either conidial hydrophobicity or zeta potential.

2.6. Data analysis

3.3. Correlations

Percent planthopper mortalities caused by the tested isolates in the bioassays were corrected with the corresponding control mortalities using Abbott’s formula. Arcsine squared roots of the corrected mortalities from each bioassay were subjected to one-way analysis of variances (ANOVA) among the isolates. For the isolates selected from in vivo passages, corrected mortalities (Mc), Pr1 activities (Ap), hydrophobicity rates (Hr) and zeta potentials (Pz) were separately compared from one passage to another (designating N for 0–3 times of in vivo passages) using the procedure of Fisher’s least significant difference (LSD) after one-way ANOVA. The variable Mc was then correlated to the variables N, Ap, Hr and Pz using the procedure of stepwise multivariate correlation in DPS software (Tang and Feng, 2007). A linear correlation analysis was also performed between paired variables except Mc.

Based on the data in Table 2, the corrected mortalities (Mc) of N. lugens caused by Bb2860, Bb2861 and Bb2879 were significantly correlated to the number of their in vivo passages (N), the conidial hydrophobicity rate (Hr), and the interactions of both and of Hr with the zeta potential (Pz), yielding Mc= 196.7 + 125.5N + 3.56Hr 1.57HrN + 0.027HrPz (r2 = 0.89, F4,7 = 13.5, P < 0.01). The significant variables (P < 0.05 in Student’s t tests for their coefficients) made an overall contribution of 89% to the mortality variation in all bioassays. The mortality increase depended mainly on the variables N and Hr based on the fitted coefficients. In linear correlation analysis, a significant positive correlation was found between Hr and N (r2 = 0.64, F1,10 = 17.7, P = 0.0018), Pz and N (r2 = 0.52, F1,10 = 10.9, P = 0.0080), N and Pr1 activity Ap (r2 = 0.51, F1,10 = 10.5, P = 0.0090), Hr and Ap (r2 = 0.45, F1,10 = 8.1, P = 0.0175), and Pz and Ap (r2 = 0.57, F1,10 = 13.2, P = 0.0046), respectively, but was absent between Hr and Pz

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Fig. 1. Effects of in vivo passages of heterologous Beauveria bassiana isolates on the corrected mortalities of Nilaparvata lugens nymphs sprayed at ca. 1000 conidia/mm2. (a) Initial bioassay prior to passing through N. lugens. (b) Bioassay after the first in vivo passage. (c) Bioassay after the second in vivo passage. Error bars: SD for the mean of three replicates.

Table 2 Changes in corrected Nilaparvata lugens mortality, conidial Pr1 activity, hydrophobicity rate, and zeta potential during three passages of three Beaueria bassiana isolates through the heterologous host insect species. Fungal isolate

a

Host passage

Mean ± SDa Mortality (%)

Pr1 activity (lM ml

1

min

1

)

Zeta potential (mV)

Hydrophobicity rate (%)

Bb2860

Initial First Second Third

17.8 ± 4.2 39.3 ± 7.1 51.4 ± 7.9 47.2 ± 5.3

c b a ab

1.25 ± 0.028 1.29 ± 0.019 1.44 ± 0.028 1.43 ± 0.051

b b a a

21.36 ± 3.09 19.37 ± 4.01 19.42 ± 4.20 18.16 ± 4.53

b ab ab a

69.72 ± 1.00 76.66 ± 1.73 78.72 ± 1.60 78.32 ± 1.77

b a a a

Bb2861

Initial First Second Third

9.4 ± 2.8 b 31.3 ± 12.6 a 44.7 ± 1.7 a 42.1 ± 7.9 a

0.80 ± 0.093 1.54 ± 0.031 1.70 ± 0.057 1.67 ± 0.033

a b c ab

29.30 ± 6.65 26.40 ± 4.98 17.89 ± 4.36 17.06 ± 1.54

b b a a

74.39 ± 3.07 78.94 ± 4.36 78.94 ± 4.36 84.59 ± 2.24

b b b a

Bb2879

Initial First Second Third

16.9 ± 7.6 c 31.1 ± 12.4 bc 62.3 ± 16.8 a 48.1 ± 7.0 ab

1.16 ± 0.106 1.60 ± 0.015 1.60 ± 0.021 1.61 ± 0.010

b a a a

24.07 ± 2.00 b 18.92 ± 2.64 a 17.36 ± 2.91 a 18.71 ± 3.23 a

70.90 ± 1.83 76.40 ± 0.85 78.07 ± 1.07 77.68 ± 4.23

b a a a

The means of each variable from each isolate were compared across the in vivo passages using Fisher’s LSD after one-way ANOVA.

(r2 = 0.22, F1,10 = 2.8, P = 0.1245). These correlations suggest some dependence (51–64%) of the conidial surface properties and Pr1 activity on the number of in vivo passages. Interestingly, Pr1 activity contributed significantly to the two surface properties independent of each other. Based on the data from four other isolates in Table 3, however, linear correlations were not detected between all paired variables except between N and Ap (r2 = 0.81, F1,10 = 43.57, P < 0.001).

4. Discussion As presented above, all 21 heterologous B. bassiana isolates showed low pathogenicity to N. lugens nymphs sprayed at the high

concentration of 1000 conidia/mm2 prior to passage through the pest species. Of those, three isolates originally derived from aphids exhibited a pathogenicity increasing with the number of in vivo passages for host adaptation. As a result, their pathogenicity to N. lugens was enhanced 3–4-fold by the first two in vivo passages but no more by further passage. This is largely in agreement with a previous report that the host-induced adaptation of M. anisopliae was essentially complete resulting in a significant increase in virulence after the first in vivo passage (Fargues and Robert, 1983). However, our data indicate that not all strains display equal host plasticity and no increased virulence was noted to several isolates even after passage through N. lugens. The heterologous host-induced adaptation of the three B. bassiana isolates in this study resulted in maximal mortalities of

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Table 3 Changes in corrected Nilaparvata lugens mortality, conidial Pr1 activity, hydrophobicity rate, and zeta potential during two passages of four Beaueria bassiana isolates through the heterologous host insect species. Fungal isolate

Bb734

Bb1269

Bb2864

Bb4535

a

Host passage

Initial First Second Initial First Second Initial First Second Initial First Second

Mean ± SDa Mortality (%)

Pr1 activity (lM ml

18.9 ± 1.7 a 19.4 ± 4.1 a 18.8 ± 4.9 a 14.1 ± 5.7 a 22.3 ± 1.9 a 25.3 ± 1.7 a 11.7 ± 0.87 a 27.5 ± 12.1 a 23.8 ± 8.5 a 20.8 ± 5.3 a 17.1 ± 10.3 a 21.2 ± 1.0 a

0.57 ± 0.018 b 1.43 ± 0.020 a 1.47 ± 0.18 a 0.59 ± 0.013 c 1.06 ± 0.010 b 1.61 ± 0.015 a 0.64 ± 0.010 b 1.49 ± 0.032 a 1.54 ± 0.021 a 0.65 ± 0.0130 c 1.15 ± 0.028 b 1.29 ± 0.047 a

1

min

1

)

Zeta potential (mV)

Hydrophobicity rate (%)

19.8 ± 3.86 a 20.7 ± 2.44 ab 22.6 ± 1.66 b 36.4 ± 3.84 c 29.7 ± 8.60 b 22.3 ± 7.82 a 32.7 ± 3.27 b 31.9 ± 2.44 b 28.5 ± 1.74 a 10.8 ± 3.35 a 5.5 ± 1.65 b 27.7 ± 3.59 c

76.87 ± 0.77 76.53 ± 1.65 72.57 ± 1.83 72.57 ± 2.09 77.51 ± 1.65 73.34 ± 1.83 86.57 ± 2.07 82.30 ± 0.74 82.42 ± 1.08 75.33 ± 1.94 81.35 ± 0.92 70.65 ± 3.93

a a a b a ab a b b ab a b

The means of each variable from each isolate were compared across the in vivo passages using Fisher’s LSD after one-way ANOVA.

45–62% under the concentrated spray. Such mortalities were similar to what were observed on the same pest species infected by two N. lugens derived M. anisopliae isolates, which were selected from 35 original or heterologous isolates of Metarhizium spp. (Jin et al., 2008), and a few potential candidates chosen from a large number of fungal isolates (Roberts and St. Leger, 2004). A single isolate selected from 17 B. bassiana isolates also showed similar virulence to the hopper species Peregrinus maidis, Delphacodes kuscheli and Dalbulus maidis (Toledo et al., 2007). The previous reports and our study witnessed the same challenge for screening a fungal candidate to kill more than 70% planthoppers or leafhoppers through routine bioassays. The increase of fungal pathogenicity by the heterologous hostinduced adaptation is likely attributed to improved conidial adhesion to insect cuticle because the elevation of both conidial hydrophobicity and zeta potential may result in the improvement (Boucias et al., 1988; Holder and Keyhani, 2005; Cho et al., 2007). Such surface properties could be potential indices for the success of the heterologous host-induced adaptation. Apart from the well-known role of conidial hydrophobicity in cuticle adhesion, particles with zeta potentials more positive than +30 mV or more negative than 30 mV are normally considered stable (http:// www.silver-colloids.com/Tutorials/Intro/pcs12.html). In our study, conidial zeta potentials of the three isolates in Table 2 were closer to 30 mV prior to the first in vivo passage but departed significantly from the initial levels after one or two passages through N. lugens. The conidia at more unstable status could adapt more readily to the heterologous insect species, as evidenced by the significant increases of their hydrophobicity rates to favor cuticle adhesion for more successful infection. Interestingly, the conidial Pr1 activities of the three isolates adapted to the heterologous insect species were positively correlated to the two surface properties. This supports previous observations on the Pr1 virulence factor and the surface properties (Wang et al., 2002; Shah et al., 2007). The surface properties and Pr1 activities improved by the in vivo passages may result from enhanced expression levels of related genes existing in the isolates. However, the host adaptation may not change the isolates genetically. Perhaps for this reason, it is not surprising to see no genetic change in fungal strains with pathogenic or phenotypic features altered by serial transfers (Vandenberg and Cantone, 2004). Finally, not all heterologous isolates enable to adapt to an insect species by repeated in vivo passages. The four isolates in Table 3 had no significant change in pathogenicity after two passages through N. lugens. Previously, two M. anisopliae pathotypes, specific respectively for Cetonia aurata and Oryctes rhinoceros, were not modified in their pathogenicity to a nonsusceptible species by

passing through the respective insect hosts (Fargues and Robert, 1983). Most importantly, we found that the surface properties of conidial hydrophobicity and zeta potential associated with cuticle adhesion and pathogenicity (Boucias et al., 1988; Holder and Keyhani, 2005; Cho et al., 2007) are useful indices for the success of fungal adaptation to the heterologous host insect species. This finding would help to understand fungus-insect interactions and to find more potential fungal candidates against insect pests, such as N. lugens, by means of sequential in vivo passages. Acknowledgements We are grateful to R.A. Humber (RW Holley Center for Agriculture and Health, Ithaca, NY, USA) for providing ARSEF isolates. Funding of this study was provided jointly by the Natural Science Foundation of China (30930018), the Ministry of Science and Technology (2009CB118904), and the Zhejiang R&D program (2009R50G2010001). References Backus, E.A., Serrano, M.S., Ranger, C.M., 2005. Mechanisms of hopperburn: an overview of insect taxonomy, behavior, and physiology. Annu. Rev. Entomol. 50, 125–151. Bernal, C.C., Aguda, R.M., Cohen, M.B., 2002. Effect of rice lines transformed with Bacillus thuringiensis toxin genes on the brown planthopper and its predator Cyrtorhinus lividipennis. Entomol. Exp. Appl. 102, 21–28. Boucias, D.G., Pendland, J.C., Latge, J.P., 1988. Nonspecific factors involved in the attachment of entomopathogenic deuteromycetes to host insect cuticle. Appl. Environ. Microbiol. 54, 1797–1805. Brownbridge, M., Costa, S., Jaronski, S.T., 2001. Effects of in vitro passage of Beauveria bassiana on virulence to Bemisia argentifolii. J. Invertebr. Pathol. 77, 280–283. Cho, E.M., Kirkland, B.H., Holder, D.J., Keyhani, N.O., 2007. Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Microbiology – SGM 153, 3438–3447. Clarkson, J.M., Charnley, A.K., 1996. New insights into the mechanisms of fungal pathogenesis in insects. Trends Microbiol. 4, 197–203. Dias, B.A., Neves, P.M.O.J., Furlaneto-Maia, L., Furlaneto, M.C., 2008. Cuticledegrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence of coffee berry borer cuticle. Braz. J. Mircrobiol. 39, 301–306. Fargues, J.F., Robert, P.H., 1983. Effect of passaging through scarabeid hosts on the virulence and host specificity of two strains of the entomopathogenic hyphomycete Metarhizium anisopliae. Can. J. Microbiol. 29, 576–583. Feng, M.G., Poprawski, T.J., Khachatourians, G.G., 1994. Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: current status. Biocontrol Sci. Technol. 4, 3–34. Feng, M.G., Pu, X.Y., 2005. Time-concentration-mortality modeling of the synergistic interaction of Beauveria bassiana and imidacloprid against Nilaparvata lugens. Pest Manag. Sci. 61, 363–370. Freimoser, F.M., Screen, S., Bagga, S., Hu, G., St. Leger, R.J., 2003. Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts. Microbiology – SGM 149, 239–247.

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