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Differential fluctuation in virulence and VOC profiles among different cultures of entomopathogenic fungi
Journal of Invertebrate Pathology 104 (2010) 166–171
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Differential fluctuation in virulence and VOC profiles among different cultures of entomopathogenic fungi Abid Hussain *, Ming-Yi Tian, Yu-Rong He, Yan-Yuan Lei Department of Entomology, College of Natural Resources and Environment, South China Agricultural University, Guangzhou, 510640, China
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Article history: Received 11 November 2009 Accepted 12 March 2010 Available online 15 March 2010 Keywords: Beauveria bassiana Coptotermes formosanus HS-SPME/GC–MS Metarhizium anisopliae n-tetradecane Virulence Volatiles
a b s t r a c t Insect-passaged cultures of entomopathogenic fungi grown on potato dextrose agar media have been shown to have altered virulence and profiles of volatile compounds. The present study demonstrated the pathogenic status of FS0 (in vitro) and FS1 and FS2 (insect-passaged cultures grown on PDA) cultures of Metarhizium anisopliae (strains 406 and 02049) and Beauveria bassiana by a non-choice assay, in which filter paper was inoculated with fungal spores at a concentration of 1 107 spores/ml. The FS1 and FS2 cultures of M. anisopliae strain 02049 and B. bassiana produced conidia with high virulence, and the volatile profiles of these conidia comprised relatively lower percentages of branched-alkanes than conidia from the FS0 cultures. In contrast, the conidia from an FS0 culture of M. anisopliae strain 406 had somewhat elevated virulence levels, but their volatile profile had <2% branched-alkanes. The FS1 and FS2 cultures of M. anisopliae strain 406 did not gain virulence, and these cultures showed a decline in virulence along with major alteration of their volatile profiles. Their volatile profiles mainly comprised branchedalkanes. The volatile profiles of the FS1 and FS2 cultures lacked n-tetradecane, which was an important component of all the virulent cultures. Four compounds, 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane, were detected only from the virulent cultures, suggesting that low LT50 values were probably due to the production of these compounds. This is the first report to characterize volatiles from FS0, FS1 and FS2 cultures of entomopathogenic fungi; its utility in different aspects opens an interesting area for further investigations. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction In accordance with increasing global concern for the protection of public health and the environment, in Mainland China there is growing public and governmental pressure to minimize the use of synthetic pesticides for the management of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Consequently, alternative methods to control termites are urgently needed. Experiments during the last few decades with Metarhizium and Beauveria, fungal pathogens of termites, have shown great promise for commercial development because these fungi are distributed worldwide in the soils termites usually inhabit (Wells et al., 1995). Many isolates of these fungi are virulent to termites under laboratory conditions (Milner et al., 1998). The stability of virulence following in vitro passage is a desirable trait for the production of mycoinsecticides (Vandenberg and Cantone, 2004). However, the successive subculturing of entomopathogenic fungi on artificial culture media may lead to instability and attenuation of fungal cultures (Vandenberg and Cantone, 2004; Shah et al., 2007). In the past, a number of investigations have been * Corresponding author. E-mail address: [email protected] (A. Hussain). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.03.004
conducted with the objective of restoring the virulence of the strains. Crespo et al. (2002) compared the performance of Beauveria bassiana grown on a complete agar medium containing glucose as the carbon source with that grown on alkane (n-hexadecane) medium. They found a significant increase in the mortality of Acanthoscelides obtectus grown on alkane culture medium. The early findings of Shah et al. (2005) clearly describe that nutrition influences growth, sporulation and virulence of the insect pathogenic fungus, Metarhizium anisopliae. Furthermore, they suggested that Tenebrio molitor larvae are more susceptible to the conidia produced on 1% yeast extract, 2% peptone osmotic stress medium (OSM), and CN 10:1 medium as well as conidia cultivated from susceptible host (Galleria mellonela and T. molitor) larvae. The effects of serial passage, whether in vitro or in vivo, on spore size, germination, attachment to the cuticle (Altre et al., 1999) and the activity of spore-bound Pr1 (Shah et al., 2007) remained the subject of many investigations. They suggested that the attenuated cultures undergo an alteration of growth rate, reduced sporulation, and the formation of sectors with a decline in virulence. In addition, the entomopathogenic fungi produce cuticle-degrading enzymes, including esterases, lipases, N-acetylglucosaminidases and chitinases, thought to be utilized in degradation of the host’s cuticle (St. Leger et al., 1986). The investigations of Lecuona et al.
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(1991) documented the changes in insect epicuticular hydrocarbons of Ostrinia nubilalis and Melolontha melolontha after treatment with B. bassiana. However, relatively little attention has focused on the odor profiles of entomopathogenic fungi, and to our knowledge no investigation has profiled the volatile organic compounds (VOCs) of entomopathogenic fungi cultivated from C. formosanus. This study provides the first detailed analysis of the relationship between virulence and the VOC profiles of entomopathogenic fungi produced in vitro and in vivo. 2. Materials and methods 2.1. Termite collection and maintenance C formosanus Shiraki were collected from the forest of South China Agricultural University Guangzhou, China. Termites were maintained at 26–28 °C in plastic buckets containing pine wood stakes (Pinus spp.) placed over moist sterile soil in complete darkness. 2.2. Fungal strains The entomopathogenic fungi M. anisopliae (EBCL 02049 and 406) and B. bassiana (EBCL 03005) were originally isolated from C. formosanus in China, except for strain 406, which was isolated from soil. The strains were cultured on potato dextrose agar (200 g potato/l, 20 g/l dextrose and 20 g/l agar) at 26 ± 1 °C, in complete darkness. 2.3. Extraction and analysis of VOCs released by entomopathogenic fungi To observe the differences in the VOC profiles of conidia of entomopathogenic fungi produced in vitro and in vivo, both cultures were grown on PDA. Cultures successively grown on 20-ml headspace PDA vials at 25 ± 2 °C for 24 days were designated as FS0. Two different cultures inoculated on 20-ml headspace PDA slants cultivated from two different abdominal segments of the same host (C. formosanus) were designated as FS1 and FS2 in order to observe the variation between them. FS1 and FS2 cultures were harvested from single termite worker cadaver under microscope previously infected with FS0 cultures of M. anisopliae (EBCL 02049 and 406) and B. bassiana (EBCL 03005). These spores were then inoculated on PDA slants at 25 ± 2 °C for 24 days. Only the pure cultures were used for further experimentation. Fungal cultures were identified based on the microscopic characteristics of the studied strains. The VOCs in the air spaces of the FS0, FS1, and FS2 cultures of entomopathogenic fungi M. anisopliae (EBCL 02049 and 406) and B. bassiana (EBCL 03005) were extracted from the 20-ml headspace vials by equilibrating the cultures for 30 min at room temperature. Subsequently, a stainless needle, within which a 75-lm Carboxen/ Polydimethylsiloxane (CAR/PDMS) solid-phase microextraction (SPME) fiber (Supelco, Bellefonte, PA, USA) was housed, was pushed through the vial septum, allowing the fiber to be exposed to the headspace of the fungal cultures for 30 min at room temperature. After extraction, the SPME device was removed from the culture vial and immediately inserted into the injection port (splitless mode) of the gas chromatography–mass spectrometer (GC–MS) for thermal desorption. The control vials contained similar amounts of PDA. The peaks observed in the control (PDA) were deleted from the samples and the proportion of total remaining peaks set to 100%. The entire experiment was conducted twice. Volatile compounds were analyzed with a Finnigan Trace GC– MS (Finnigan, Boston, MA, USA) equipped with HP-1 and fused
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with a silicon capillary column (30 m 0.25 mm ID, 1 lm film thickness). Helium (99.99% purity) was used as a carrier gas with a constant flow of 1.0 ml/min. After desorption, the oven was held at 45 °C for 3 min, and then the temperature was raised to 80 °C at a rate of 5 °C/min, to 150 °C at a rate of 20 °C/min for 5 min and finally to 250 °C at the rate of 30 °C /min and held at 250 °C for 5 min. Mass spectra were repetitively scanned from 35 to 335 amu. Ionization was elected in the electron impact mode (EI) at 70 eV. Volatile compound identifications were made by comparison to spectra in the Wiley275.1 and NIST98 (National Institute of Standards and Technology, Gaithersburg, MD, USA) libraries. Some of the peaks were confirmed by comparison with peak retention times and mass spectra of purchased standards. 2.4. Comparative virulence of different cultures of entomopathogenic fungi against C. formosanus Shiraki by the non-choice assay The same slants of the above mentioned cultures of entomopathogenic fungi were used to determine the level of virulence. Twenty-four-day-old sporulating cultures of M. anisopliae and B. bassiana were harvested with 0.05% Tween 80 (Sigma). The spore germination was determined as previously described by Hussain et al. (2009). All of the strains showed >93% conidial germination. One millimeter of the conidial suspensions of FS0, FS1, and FS2 cultures of M. anisopliae and B. bassiana at a concentration of 1 107 conidia/ml were evenly distributed onto moist Whatman No. 1 filter paper with a pipette (controls were treated with this solution without conidia). One hundred termite workers of C. formosanus were added to each Petri dish (95 15 mm). The experimental units were placed at 26–28 °C in complete darkness. Dead and weak termites were replaced with healthy termites immediately before the experiment. Each treatment comprised five replicates. The entire experiment was conducted twice. Experimental plates were monitored daily. To know whether termites died from fungal infection, insect cadavers were counted and removed to allow growth of the associated microbes on the cadavers. To correct for mortality in the control, Abbot’s formula (Abbott, 1925) was applied. Data were analyzed using analysis of variance (ANOVA), and means were compared by Tukey’s HSD test. The analyses were run using the SAS system software 8.01 (2000). 3. Results 3.1. Infectivity of entomopathogenic fungi The virulence of the FS0 cultures of the entomopathogenic fungi M. anisopliae strains 406 and 02049 and B. bassiana against C. formosanus in Petri dishes differed significantly (F = 48.99; df = 2; P < 0.001) when the filter paper was treated with conidial suspension at 1 107 conidia/ml. All strains were able to induce 100% mortality within 12 days. Strain 406 had the shortest LT50 values (2.33 d). The LT50 values for B. bassiana were significantly higher than those of either strain of M. anisopliae (Table 1). Following passage through C. formosanus (FS1 and FS2 cultures), however, the conidia of strain 406 showed reduced virulence against C. formosanus in both cultures (FS1 and FS2). On the other hand, for strains 02049 and 03005, passage through C. formosanus in FS1 as well as FS2 cultures enhanced virulence, resulting in low LT50 values compared to the conidia from the FS0 culture (Table 1). 3.2. HS-SPME/GC–MS analysis of entomopathogenic fungi The noticeable variation in VOC production between in vitro (FS0) and in vivo (FS1 and FS2) cultures of entomopathogenic fungi may suggest a difference in their biological activities. In every
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Table 1 Time mortality response of C. formosanus workers against entomopathogenic fungal strains. Fungal species
Strain
LT50 ± SE in days FS1
FS2
M. anisopliae
406 EBCL 02049
2.33 ± 0.20c 3.64 ± 0.22b
4.03 ± 0.30a 2.01 ± 0.12b
3.73 ± 0.27b 2.21 ± 0.14c
B. bassiana
EBCL 03005
5.67 ± 0.29a
4.66 ± 0.35a
4.89 ± 0.34a
FS0
Mean lethal time values within column followed by the same letter are not significantly different by the Tukey HSD Test (P = 0.05). FS0 = In vitro culture (fungal strains successively cultured on PDA for 24 days); FS1 = 1st In vivo culture (fungal strains cultivated from C. formosanus workers then incubated on PDA for 24 days as described in Section 2); FS2 = 2nd In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days).
chromatograph, the majority of substances eluted in the time range of 12–23 min, an indication that these compounds are of light to medium molecular weights (Table 2). The HS-SPME analysis of VOCs of M. anisopliae (strain 406), investigated by GC–MS, showed the presence of 5, 6, and 10 compounds from the FS0, FS1, and FS2 cultures, respectively (Table 2). The hydrocarbons identified ranged in size between C7 and C19. The volatile profile of the FS0 culture comprised mainly n-tetradecane (42.43%) and alkenes (45.04%). However, the VOC profile of the FS1 culture mainly comprised branched chain alkanes (94.99%), and the remaining profile comprised one miscellaneous compound (Fig. 1b). The profile of the FS2 culture comprised branched-alkanes (46.80%) and alkenes (29.62%). An ester (10.07%) was detected in the volatile profile of the FS2 culture, while an aldehyde (10.79%) was detected only in the FS0 culture (Fig. 1b). In vivo passage of M. anisopliae (02049) changed its gaseous profile. The VOCs of the FS0, FS1 and FS2 cultures consisted of 8, 7, and 9 compounds, respectively (Table 2). Comparatively, the FS1 and FS2 cultures had higher proportions of paraffins (35– 49%), olefins (14–17%) and ketones (21–48%). On the other hand, the FS0 culture mainly comprised 2,3,4,4a,5,6,7,8-Octahydro1,1,4a,7-tetramethyl-1H-benzocyclohepten-7-ol (81.03%) (Fig. 1c). The volatile compounds of the FS0, FS1 and FS2 cultures of B. bassiana, listed in Table 2 comprised 9, 8, and 9 compounds, respectively. The gaseous blend from the FS0 culture of B. bassiana mainly comprised five branched-alkanes (56.15%), one paraffin (21.54%) and an ester (14.05%) (Fig. 1a), while the gaseous profile from the FS1 and FS2 cultures contained high proportions of 1,5,5,8-tetramethyl-3,7-cycloundecadien-1-ol (61.22%) and olefins (37.26%), respectively. Comparatively, a lower proportion of 1,5,5,8-tetramethyl-3,7-cycloundecadien-1-ol (6.43%) was present in the profile of the FS2 culture (Fig. 1a). An aldehyde (2-phenylpropenal) was only detected in the FS1 and FS2 cultures (Table 2). 4. Discussion The present study demonstrated that culturing of entomopathogenic fungi under different conditions resulted in qualitative differences in their VOC profiles, with corresponding changes in their virulence. The pattern of change was dependent upon the strain. The conidia from insect-passaged inocula were pronouncedly virulent, suggesting that the host clearly provides the nutrition for the production of virulent conidia. However, the decline in virulence of insect-passaged inocula of M. anisopliae (406) was erratic, suggesting that the strain was at its full insecticidal potential and did not become attenuated in vitro. Evidence for a decline in virulence after in vivo culturing is a novel finding. In the past, there have been several reports that passaging does not enhance virulence because
fungal strains have undergone irreversible physiological changes and did not become attenuated through successive subculturing on nutrient media (Hall, 1980; Brownbridge et al., 2001; Vandenberg and Cantone, 2004). Furthermore, it is unclear how the FS1 and FS2 cultures of M. anisopliae (406) declined in virulence; however, this research documents that these cultures produce different volatile profiles. The VOC profiles of the FS1 and FS2 cultures mainly comprised branched-alkanes, while the virulent inocula of the FS0 culture comprised n-tetradecane (42.43%) and alkene (45.04%). The results obtained with this strain confirm previous work demonstrating variability in the virulence of B. bassiana grown on a glucose and straight-chain alkane medium. The mortality of A. obtectus increased from 22 ± 4.5% to 44 ± 11.4% on day 7 and from 26 ± 5.5% to 60 ± 7.1% on day 14 after treatment with conidia grown on glucose and n-hexadecane, respectively. Furthermore, it has been suggested that alkane supplementation of the culture medium might improve the virulence of some mycoinsecticides (Crespo et al., 2002). HS-SPME analyses of volatile compounds have revealed an impressive diversity of chemical components among cultures of entomopathogenic fungi. The volatile profiles vary among the analyzed strains and their cultures. Four compounds, 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane, were detected in varying proportions only for the highly virulent cultures of all the studied strains. However, 2,7,10-terimethyldodecane and 2,6,11-trimethyldodecane were detected in the FS1 and FS2 cultures of B. bassiana and M. anisopliae 02049. On the other hand, 2,3,7-trimethyloctane was detected but did not constitute more than 3% of the VOCs from the FS1 and FS2 cultures of M. anisopliae 406 and the FS0 cultures of B. bassiana and M. anisopliae 02049. In the bioassays, these cultures displayed declines in virulence. Similarly, 2,3,5-trimethyldecane was profiled from the FS0 culture of B. bassiana and the FS1 and FS2 cultures of M. anisopliae 406 (Table 2). Vandenberg and Cantone (2004) found that there was no change in the banding pattern of any of the studied strains before and after passing through the host. However, the complete change in the gaseous profile that was observed in the present study enables us to speculate that the variability in the gaseous profile after passing through a host results in changes in the infectivity of the tested strains. Furthermore, our findings suggest that the proportions and profile of VOCs in entomopathogenic fungi vary not only among strains but also among cultures (FS0, FS1, and FS2), which might because of nutritional status of the culture. The current results are likely to be useful in developing a medium based on these chemicals for a broad range of entomopathogenic fungi, since many entomopathogenic fungi infect several termite species in different geographical zones. The noticeable variation in VOC production of M. anisopliae (02049), in contrast to the original culture (FS0), suggests that the biological activities of these cultures may be different. The present findings of low LT50 values of the conidia of FS1 and FS2 cultures of M. anisopliae (02049) clearly indicate an increase in virulence. The in vitro subculturing of entomopathogenic fungi on artificial growth media results in rapid changes in the conidia, corresponding with a decline in virulence (Shah et al., 2007). In general, the most virulent strains are those derived from the target host (Glare and Milner, 1991). Similarly, the results obtained by Hayden et al. (1992) are in accordance with our finding that a single passage through the host enhanced the virulence of entomopathogenic fungi. Some earlier studies also strengthened our findings and suggested that virulence is restored irrespective of whether the culture is passed through the highly susceptible larvae of the wax moth (Galleria mellonella) or the flour beetle (T. molitor) (Shah et al., 2005). The enhancement of virulence is well documented,
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A. Hussain et al. / Journal of Invertebrate Pathology 104 (2010) 166–171 Table 2 Volatile organic compound profiles of entomopathogenic fungi. Fungal strain
Rt
B. bassiana (EBCL 03005)
12.74 13.36 13.48 13.74 15.34 15.85 15.94 17.11 18.33 18.79 19.10 19.16 19.42 19.60 19.86 20.41 20.92 22.25
1,4-Dimethoxybenzenea 1,2-Benzothiazole-3-carboxylic acid 2,3,6-Trimethylheptane Hexyl n-valerate 2,3,7-Trimethyloctane 2,3,5-Trimethyldecane 2-Phenylpropenal 2,2,8-Trimethyldecane 1-Propylcyclohexane 2,5,5-Trimethyl-1-hexene 2,6,8-Trimethyldecane Trifluoroacetyl-isopulegol n-Tetradecanea n-Hexadecanea 1,5,5,8-Tetramethyl-3,7-cycloundecadien-1-ol 2,7,10-Terimethyldodecane 2,6,11-Trimethyldodecane 2,6-Dimethylheptadecane
13.02 13.35 13.61 13.74 14.52 15.34 15.86 15.94 17.74 18.79 18.93 19.11 19.22 19.42 19.60 20.17 21.28 21.29 22.25
1-Methylene-1H-indene 1,2-Benzisothiazole Azulenea Hexyl n-valerate 4,7-Dimethylundecane 2,3,7-Trimethyloctane 2,3,5-Trimethyldecane 2-Phenylpropenal 2-Methyl-5-propylnonane 2,5,5-Trimethyl-1-hexene 3,7-Dimethylundecane Tridecyliodide 3-Methylpentadecanea n-Tetradecanea n-Hexadecanea 4-Isopropyl-1,6-dimethylnaphthalene 2,2,4,4,6,8,8-Heptamethylnonane 7-Methylpentadecane 2,6-Dimethylheptadecane
13.02 13.46 13.60 15.34 15.94 16.10 17.11 18.79 18.84 19.42 19.51 20.41 20.67 20.79 20.92 21.29 22.25
1-Methylene-1H-indene 4-Methylnonanea Azulenea 2,3,7-Trimethyloctane 2-Phenylpropenal 2,3,4,4a,5,6,7,8-Octahydro-1,1,4a,7-tetramethyl-1H-benzocyclohepten-7-ol 2,2,8-Trimethyldecane 2,5,5-Trimethyl-1-hexene 1-Cyclohexyloctane n-Tetradecanea 5,6,6-Trimethylundeca-3,4-diene-2,10-dione 2,7,10-Trimethyldodecane 5-Propyltridecane 2,4-ditert-butyl-6-methylphenola 2,6,11-Trimethyldodecane 7-Methylpentadecane 2,6-Dimethylheptadecane
(min)
Compounds
RA (%) FS0
M. anisopliae (406)
M. anisopliae (EBCL 02049)
FS1
FS2
8.51
17.42
12.13
1.05 37.26
5.42 8.14
4.37 20.28
61.22 0.53 2.99 1.06
6.43 2.24 8.32 2.63
1.62 6.65 1.99 14.05 2.24 42.76 4.32
4.85
21.54
8.42 5.01 8.22 10.07 14.48 9.20
2.92 14.3
69.74
19.23
0.88
2.75 6.09 5.28
10.79 36.82
42.43 7.40 21.2 0.68 2.32 1.75 2.89 0.76 1.68
3.81
1.38
6.37
12.00
12.97
35.96 47.42 0.20
48.06 21.08 0.62
1.36
1.53 4.40
1.43 81.03 2.71 5.68
2.31
3.19 1.16
Rt = Retention time; RA = Relative area (peak area relative to total peak area); FS0 = In vitro culture (fungal strains successively cultured on PDA for 24 days); FS1 = 1st In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days as described in Section 2); FS2 = 2nd In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days). a Compounds identified by authentic standards.
but very little is known about the underlying mechanisms of virulence. The chromatographic fingerprints of the FS1 and FS2 cultures of M. anisopliae (02049) changed drastically to a profile with a straight-chain alkane (n-tetradecane) and 5,6,6-trimethylundeca3,4-diene-2,10-dione as the major compounds. Comparatively, the VOC profile of a less virulent inocula of the FS0 culture lacked these hydrocarbons and mainly comprised 2,3,4,4a,5,6,7,8-Octahydro-1,1,4a,7-tetramethyl-1H-benzocyclohepten-7-ol, alkenes, branched and cyclic alkanes. On the other hand, the volatile pro-
files of restored cultures of FS1 and FS2 of B. bassiana contained n-tetradecane, while the profile of the FS0 culture lacked this hydrocarbon and contained n-hexadecane. On the whole, all of the assays indicated the significant superiority of M. anisopliae over B. bassiana in terms of effectiveness in controlling C. formosanus workers, which is consistent with previous work reported by Jones et al. (1996) and Delate et al. (1995). The low LT50 values might be due to the production of 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane. These compounds may cause modifications in termite foraging behavior; if
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Fig. 1. Main fractions of volatile compounds Linear alkanes, branched-alkanes, Cyclic alkanes, Alkenes, Alcohols, Esters, Aldehydes, Ketones, Others (percentage of total peak areas) extracted from (a) B. bassiana, (b) M. anisopliae (406), (c) M. anisopliae (EBCL 02049) cultures [FS0 = In vitro culture (fungal strains successively cultured on PDA for 24 day); FS1 = 1st In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days); FS2 = 2nd In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days)]; using HS-SPME/GC–MS.
so, it becomes reasonable to hypothesize a new biological role for VOCs as a repellent to termites that would otherwise destroy the colony by natural fungal epizootics. Certainly, this concept is worthy of further study by directly testing the foraging behavior of termites upon direct exposure to fungal cultures. On the basis of the above findings, we may conclude that the difference in virulence may arise because of the change in the hydrocarbon profile. Normally, the VOC profiles of the virulent
inocula of the studied strains comprised mainly 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane. When the percentage of branched-alkanes in the volatile profile increased, the virulence decreased. However, further research is necessary to elucidate the mechanism responsible for this decrease in virulence. The incorporation of these prominent hydrocarbons into the growth media in order to observe their effects on the infectivity of entomopathogenic fungi will be the logical
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extension of this work in order to suggest a possible approach for mycoinsecticide improvement.
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enhancement of virulence with host passage of Paecilomyces farinosus. J. Econ. Entomol. 85, 58–64. Hussain, A., Tian, M.Y., He, Y.R., Ahmed, S., 2009. Entomopathogenic fungi disturbed the larval growth and feeding performance of Ocinara varians Walker (Lepidoptera: Bombycidae) larvae. Insect Sci. 16, 511–517. Jones, W.E., Grace, J.K., Tamashiro, M., 1996. Virulence of seven isolates of Beauveria Bassiana and Metarhizium anisopliae to Coptotermes formosanus. Environ. Entomol. 25 (2), 481–487. Lecuona, R., Riba, G., Cassier, P., Clement, J.L., 1991. Alterations of insect epicuticular hydrocarbons during infection with Beauveria bassiana or B. brongniartii. J. Invertebr. Pathol. 58 (1), 10–18. Milner, R.J., Staples, J.A., Hartley, T.R., Lutton, G.G., Driver, F., Watson, J.A.L., 1998. Occurrence of the entomopathogenic fungus, Metarhizium anisopliae, in nests and feeding sites of Australian termites. Mycol. Res. 102, 216–220. SAS Institute, 2000. SAS User’s Guide: Statistics. SAS Institute, Cary, New York. Shah, F.A., Wang, C.S., Butt, T.M., 2005. Nutrition influences growth and virulence of the insect-pathogenic fungus Metarhizium anisopliae. FEMS Microbiol. Lett. 251, 259–266. Shah, F.A., Allen, N., Wright, C.J., Butt, T.M., 2007. Repeated in vitro subculturing alters spore surface properties and virulence of Metarhizium anisopliae. FEMS Microbiol. Lett. 276, 60–66. St. Leger, R.J., Charnley, A.K., Cooper, R.M., 1986. Cuticle degrading enzymes of entomopathogenic fungi: synthesis in culture on cuticle. J. Invertebr. Pathol. 48, 85–95. Vandenberg, J.D., Cantone, F.A., 2004. Effect of serial transfer of three strains of Paecilomyces fumosoroseus on growth in vitro, virulence and host specificity. J. Invertebr. Pathol. 85 (1), 40–45. Wells, J.D., Fuxa, J.R., Henderson, G., 1995. Virulence of four fungal pathogens to Coptotermes formosanus (Isoptera: Rhinotermitidae). J. Entomol. Sci. 30, 208–215.
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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Differential fluctuation in virulence and VOC profiles among different cultures of entomopathogenic fungi Abid Hussain *, Ming-Yi Tian, Yu-Rong He, Yan-Yuan Lei Department of Entomology, College of Natural Resources and Environment, South China Agricultural University, Guangzhou, 510640, China
a r t i c l e
i n f o
Article history: Received 11 November 2009 Accepted 12 March 2010 Available online 15 March 2010 Keywords: Beauveria bassiana Coptotermes formosanus HS-SPME/GC–MS Metarhizium anisopliae n-tetradecane Virulence Volatiles
a b s t r a c t Insect-passaged cultures of entomopathogenic fungi grown on potato dextrose agar media have been shown to have altered virulence and profiles of volatile compounds. The present study demonstrated the pathogenic status of FS0 (in vitro) and FS1 and FS2 (insect-passaged cultures grown on PDA) cultures of Metarhizium anisopliae (strains 406 and 02049) and Beauveria bassiana by a non-choice assay, in which filter paper was inoculated with fungal spores at a concentration of 1 107 spores/ml. The FS1 and FS2 cultures of M. anisopliae strain 02049 and B. bassiana produced conidia with high virulence, and the volatile profiles of these conidia comprised relatively lower percentages of branched-alkanes than conidia from the FS0 cultures. In contrast, the conidia from an FS0 culture of M. anisopliae strain 406 had somewhat elevated virulence levels, but their volatile profile had <2% branched-alkanes. The FS1 and FS2 cultures of M. anisopliae strain 406 did not gain virulence, and these cultures showed a decline in virulence along with major alteration of their volatile profiles. Their volatile profiles mainly comprised branchedalkanes. The volatile profiles of the FS1 and FS2 cultures lacked n-tetradecane, which was an important component of all the virulent cultures. Four compounds, 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane, were detected only from the virulent cultures, suggesting that low LT50 values were probably due to the production of these compounds. This is the first report to characterize volatiles from FS0, FS1 and FS2 cultures of entomopathogenic fungi; its utility in different aspects opens an interesting area for further investigations. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction In accordance with increasing global concern for the protection of public health and the environment, in Mainland China there is growing public and governmental pressure to minimize the use of synthetic pesticides for the management of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Consequently, alternative methods to control termites are urgently needed. Experiments during the last few decades with Metarhizium and Beauveria, fungal pathogens of termites, have shown great promise for commercial development because these fungi are distributed worldwide in the soils termites usually inhabit (Wells et al., 1995). Many isolates of these fungi are virulent to termites under laboratory conditions (Milner et al., 1998). The stability of virulence following in vitro passage is a desirable trait for the production of mycoinsecticides (Vandenberg and Cantone, 2004). However, the successive subculturing of entomopathogenic fungi on artificial culture media may lead to instability and attenuation of fungal cultures (Vandenberg and Cantone, 2004; Shah et al., 2007). In the past, a number of investigations have been * Corresponding author. E-mail address: [email protected] (A. Hussain). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.03.004
conducted with the objective of restoring the virulence of the strains. Crespo et al. (2002) compared the performance of Beauveria bassiana grown on a complete agar medium containing glucose as the carbon source with that grown on alkane (n-hexadecane) medium. They found a significant increase in the mortality of Acanthoscelides obtectus grown on alkane culture medium. The early findings of Shah et al. (2005) clearly describe that nutrition influences growth, sporulation and virulence of the insect pathogenic fungus, Metarhizium anisopliae. Furthermore, they suggested that Tenebrio molitor larvae are more susceptible to the conidia produced on 1% yeast extract, 2% peptone osmotic stress medium (OSM), and CN 10:1 medium as well as conidia cultivated from susceptible host (Galleria mellonela and T. molitor) larvae. The effects of serial passage, whether in vitro or in vivo, on spore size, germination, attachment to the cuticle (Altre et al., 1999) and the activity of spore-bound Pr1 (Shah et al., 2007) remained the subject of many investigations. They suggested that the attenuated cultures undergo an alteration of growth rate, reduced sporulation, and the formation of sectors with a decline in virulence. In addition, the entomopathogenic fungi produce cuticle-degrading enzymes, including esterases, lipases, N-acetylglucosaminidases and chitinases, thought to be utilized in degradation of the host’s cuticle (St. Leger et al., 1986). The investigations of Lecuona et al.
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(1991) documented the changes in insect epicuticular hydrocarbons of Ostrinia nubilalis and Melolontha melolontha after treatment with B. bassiana. However, relatively little attention has focused on the odor profiles of entomopathogenic fungi, and to our knowledge no investigation has profiled the volatile organic compounds (VOCs) of entomopathogenic fungi cultivated from C. formosanus. This study provides the first detailed analysis of the relationship between virulence and the VOC profiles of entomopathogenic fungi produced in vitro and in vivo. 2. Materials and methods 2.1. Termite collection and maintenance C formosanus Shiraki were collected from the forest of South China Agricultural University Guangzhou, China. Termites were maintained at 26–28 °C in plastic buckets containing pine wood stakes (Pinus spp.) placed over moist sterile soil in complete darkness. 2.2. Fungal strains The entomopathogenic fungi M. anisopliae (EBCL 02049 and 406) and B. bassiana (EBCL 03005) were originally isolated from C. formosanus in China, except for strain 406, which was isolated from soil. The strains were cultured on potato dextrose agar (200 g potato/l, 20 g/l dextrose and 20 g/l agar) at 26 ± 1 °C, in complete darkness. 2.3. Extraction and analysis of VOCs released by entomopathogenic fungi To observe the differences in the VOC profiles of conidia of entomopathogenic fungi produced in vitro and in vivo, both cultures were grown on PDA. Cultures successively grown on 20-ml headspace PDA vials at 25 ± 2 °C for 24 days were designated as FS0. Two different cultures inoculated on 20-ml headspace PDA slants cultivated from two different abdominal segments of the same host (C. formosanus) were designated as FS1 and FS2 in order to observe the variation between them. FS1 and FS2 cultures were harvested from single termite worker cadaver under microscope previously infected with FS0 cultures of M. anisopliae (EBCL 02049 and 406) and B. bassiana (EBCL 03005). These spores were then inoculated on PDA slants at 25 ± 2 °C for 24 days. Only the pure cultures were used for further experimentation. Fungal cultures were identified based on the microscopic characteristics of the studied strains. The VOCs in the air spaces of the FS0, FS1, and FS2 cultures of entomopathogenic fungi M. anisopliae (EBCL 02049 and 406) and B. bassiana (EBCL 03005) were extracted from the 20-ml headspace vials by equilibrating the cultures for 30 min at room temperature. Subsequently, a stainless needle, within which a 75-lm Carboxen/ Polydimethylsiloxane (CAR/PDMS) solid-phase microextraction (SPME) fiber (Supelco, Bellefonte, PA, USA) was housed, was pushed through the vial septum, allowing the fiber to be exposed to the headspace of the fungal cultures for 30 min at room temperature. After extraction, the SPME device was removed from the culture vial and immediately inserted into the injection port (splitless mode) of the gas chromatography–mass spectrometer (GC–MS) for thermal desorption. The control vials contained similar amounts of PDA. The peaks observed in the control (PDA) were deleted from the samples and the proportion of total remaining peaks set to 100%. The entire experiment was conducted twice. Volatile compounds were analyzed with a Finnigan Trace GC– MS (Finnigan, Boston, MA, USA) equipped with HP-1 and fused
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with a silicon capillary column (30 m 0.25 mm ID, 1 lm film thickness). Helium (99.99% purity) was used as a carrier gas with a constant flow of 1.0 ml/min. After desorption, the oven was held at 45 °C for 3 min, and then the temperature was raised to 80 °C at a rate of 5 °C/min, to 150 °C at a rate of 20 °C/min for 5 min and finally to 250 °C at the rate of 30 °C /min and held at 250 °C for 5 min. Mass spectra were repetitively scanned from 35 to 335 amu. Ionization was elected in the electron impact mode (EI) at 70 eV. Volatile compound identifications were made by comparison to spectra in the Wiley275.1 and NIST98 (National Institute of Standards and Technology, Gaithersburg, MD, USA) libraries. Some of the peaks were confirmed by comparison with peak retention times and mass spectra of purchased standards. 2.4. Comparative virulence of different cultures of entomopathogenic fungi against C. formosanus Shiraki by the non-choice assay The same slants of the above mentioned cultures of entomopathogenic fungi were used to determine the level of virulence. Twenty-four-day-old sporulating cultures of M. anisopliae and B. bassiana were harvested with 0.05% Tween 80 (Sigma). The spore germination was determined as previously described by Hussain et al. (2009). All of the strains showed >93% conidial germination. One millimeter of the conidial suspensions of FS0, FS1, and FS2 cultures of M. anisopliae and B. bassiana at a concentration of 1 107 conidia/ml were evenly distributed onto moist Whatman No. 1 filter paper with a pipette (controls were treated with this solution without conidia). One hundred termite workers of C. formosanus were added to each Petri dish (95 15 mm). The experimental units were placed at 26–28 °C in complete darkness. Dead and weak termites were replaced with healthy termites immediately before the experiment. Each treatment comprised five replicates. The entire experiment was conducted twice. Experimental plates were monitored daily. To know whether termites died from fungal infection, insect cadavers were counted and removed to allow growth of the associated microbes on the cadavers. To correct for mortality in the control, Abbot’s formula (Abbott, 1925) was applied. Data were analyzed using analysis of variance (ANOVA), and means were compared by Tukey’s HSD test. The analyses were run using the SAS system software 8.01 (2000). 3. Results 3.1. Infectivity of entomopathogenic fungi The virulence of the FS0 cultures of the entomopathogenic fungi M. anisopliae strains 406 and 02049 and B. bassiana against C. formosanus in Petri dishes differed significantly (F = 48.99; df = 2; P < 0.001) when the filter paper was treated with conidial suspension at 1 107 conidia/ml. All strains were able to induce 100% mortality within 12 days. Strain 406 had the shortest LT50 values (2.33 d). The LT50 values for B. bassiana were significantly higher than those of either strain of M. anisopliae (Table 1). Following passage through C. formosanus (FS1 and FS2 cultures), however, the conidia of strain 406 showed reduced virulence against C. formosanus in both cultures (FS1 and FS2). On the other hand, for strains 02049 and 03005, passage through C. formosanus in FS1 as well as FS2 cultures enhanced virulence, resulting in low LT50 values compared to the conidia from the FS0 culture (Table 1). 3.2. HS-SPME/GC–MS analysis of entomopathogenic fungi The noticeable variation in VOC production between in vitro (FS0) and in vivo (FS1 and FS2) cultures of entomopathogenic fungi may suggest a difference in their biological activities. In every
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Table 1 Time mortality response of C. formosanus workers against entomopathogenic fungal strains. Fungal species
Strain
LT50 ± SE in days FS1
FS2
M. anisopliae
406 EBCL 02049
2.33 ± 0.20c 3.64 ± 0.22b
4.03 ± 0.30a 2.01 ± 0.12b
3.73 ± 0.27b 2.21 ± 0.14c
B. bassiana
EBCL 03005
5.67 ± 0.29a
4.66 ± 0.35a
4.89 ± 0.34a
FS0
Mean lethal time values within column followed by the same letter are not significantly different by the Tukey HSD Test (P = 0.05). FS0 = In vitro culture (fungal strains successively cultured on PDA for 24 days); FS1 = 1st In vivo culture (fungal strains cultivated from C. formosanus workers then incubated on PDA for 24 days as described in Section 2); FS2 = 2nd In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days).
chromatograph, the majority of substances eluted in the time range of 12–23 min, an indication that these compounds are of light to medium molecular weights (Table 2). The HS-SPME analysis of VOCs of M. anisopliae (strain 406), investigated by GC–MS, showed the presence of 5, 6, and 10 compounds from the FS0, FS1, and FS2 cultures, respectively (Table 2). The hydrocarbons identified ranged in size between C7 and C19. The volatile profile of the FS0 culture comprised mainly n-tetradecane (42.43%) and alkenes (45.04%). However, the VOC profile of the FS1 culture mainly comprised branched chain alkanes (94.99%), and the remaining profile comprised one miscellaneous compound (Fig. 1b). The profile of the FS2 culture comprised branched-alkanes (46.80%) and alkenes (29.62%). An ester (10.07%) was detected in the volatile profile of the FS2 culture, while an aldehyde (10.79%) was detected only in the FS0 culture (Fig. 1b). In vivo passage of M. anisopliae (02049) changed its gaseous profile. The VOCs of the FS0, FS1 and FS2 cultures consisted of 8, 7, and 9 compounds, respectively (Table 2). Comparatively, the FS1 and FS2 cultures had higher proportions of paraffins (35– 49%), olefins (14–17%) and ketones (21–48%). On the other hand, the FS0 culture mainly comprised 2,3,4,4a,5,6,7,8-Octahydro1,1,4a,7-tetramethyl-1H-benzocyclohepten-7-ol (81.03%) (Fig. 1c). The volatile compounds of the FS0, FS1 and FS2 cultures of B. bassiana, listed in Table 2 comprised 9, 8, and 9 compounds, respectively. The gaseous blend from the FS0 culture of B. bassiana mainly comprised five branched-alkanes (56.15%), one paraffin (21.54%) and an ester (14.05%) (Fig. 1a), while the gaseous profile from the FS1 and FS2 cultures contained high proportions of 1,5,5,8-tetramethyl-3,7-cycloundecadien-1-ol (61.22%) and olefins (37.26%), respectively. Comparatively, a lower proportion of 1,5,5,8-tetramethyl-3,7-cycloundecadien-1-ol (6.43%) was present in the profile of the FS2 culture (Fig. 1a). An aldehyde (2-phenylpropenal) was only detected in the FS1 and FS2 cultures (Table 2). 4. Discussion The present study demonstrated that culturing of entomopathogenic fungi under different conditions resulted in qualitative differences in their VOC profiles, with corresponding changes in their virulence. The pattern of change was dependent upon the strain. The conidia from insect-passaged inocula were pronouncedly virulent, suggesting that the host clearly provides the nutrition for the production of virulent conidia. However, the decline in virulence of insect-passaged inocula of M. anisopliae (406) was erratic, suggesting that the strain was at its full insecticidal potential and did not become attenuated in vitro. Evidence for a decline in virulence after in vivo culturing is a novel finding. In the past, there have been several reports that passaging does not enhance virulence because
fungal strains have undergone irreversible physiological changes and did not become attenuated through successive subculturing on nutrient media (Hall, 1980; Brownbridge et al., 2001; Vandenberg and Cantone, 2004). Furthermore, it is unclear how the FS1 and FS2 cultures of M. anisopliae (406) declined in virulence; however, this research documents that these cultures produce different volatile profiles. The VOC profiles of the FS1 and FS2 cultures mainly comprised branched-alkanes, while the virulent inocula of the FS0 culture comprised n-tetradecane (42.43%) and alkene (45.04%). The results obtained with this strain confirm previous work demonstrating variability in the virulence of B. bassiana grown on a glucose and straight-chain alkane medium. The mortality of A. obtectus increased from 22 ± 4.5% to 44 ± 11.4% on day 7 and from 26 ± 5.5% to 60 ± 7.1% on day 14 after treatment with conidia grown on glucose and n-hexadecane, respectively. Furthermore, it has been suggested that alkane supplementation of the culture medium might improve the virulence of some mycoinsecticides (Crespo et al., 2002). HS-SPME analyses of volatile compounds have revealed an impressive diversity of chemical components among cultures of entomopathogenic fungi. The volatile profiles vary among the analyzed strains and their cultures. Four compounds, 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane, were detected in varying proportions only for the highly virulent cultures of all the studied strains. However, 2,7,10-terimethyldodecane and 2,6,11-trimethyldodecane were detected in the FS1 and FS2 cultures of B. bassiana and M. anisopliae 02049. On the other hand, 2,3,7-trimethyloctane was detected but did not constitute more than 3% of the VOCs from the FS1 and FS2 cultures of M. anisopliae 406 and the FS0 cultures of B. bassiana and M. anisopliae 02049. In the bioassays, these cultures displayed declines in virulence. Similarly, 2,3,5-trimethyldecane was profiled from the FS0 culture of B. bassiana and the FS1 and FS2 cultures of M. anisopliae 406 (Table 2). Vandenberg and Cantone (2004) found that there was no change in the banding pattern of any of the studied strains before and after passing through the host. However, the complete change in the gaseous profile that was observed in the present study enables us to speculate that the variability in the gaseous profile after passing through a host results in changes in the infectivity of the tested strains. Furthermore, our findings suggest that the proportions and profile of VOCs in entomopathogenic fungi vary not only among strains but also among cultures (FS0, FS1, and FS2), which might because of nutritional status of the culture. The current results are likely to be useful in developing a medium based on these chemicals for a broad range of entomopathogenic fungi, since many entomopathogenic fungi infect several termite species in different geographical zones. The noticeable variation in VOC production of M. anisopliae (02049), in contrast to the original culture (FS0), suggests that the biological activities of these cultures may be different. The present findings of low LT50 values of the conidia of FS1 and FS2 cultures of M. anisopliae (02049) clearly indicate an increase in virulence. The in vitro subculturing of entomopathogenic fungi on artificial growth media results in rapid changes in the conidia, corresponding with a decline in virulence (Shah et al., 2007). In general, the most virulent strains are those derived from the target host (Glare and Milner, 1991). Similarly, the results obtained by Hayden et al. (1992) are in accordance with our finding that a single passage through the host enhanced the virulence of entomopathogenic fungi. Some earlier studies also strengthened our findings and suggested that virulence is restored irrespective of whether the culture is passed through the highly susceptible larvae of the wax moth (Galleria mellonella) or the flour beetle (T. molitor) (Shah et al., 2005). The enhancement of virulence is well documented,
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A. Hussain et al. / Journal of Invertebrate Pathology 104 (2010) 166–171 Table 2 Volatile organic compound profiles of entomopathogenic fungi. Fungal strain
Rt
B. bassiana (EBCL 03005)
12.74 13.36 13.48 13.74 15.34 15.85 15.94 17.11 18.33 18.79 19.10 19.16 19.42 19.60 19.86 20.41 20.92 22.25
1,4-Dimethoxybenzenea 1,2-Benzothiazole-3-carboxylic acid 2,3,6-Trimethylheptane Hexyl n-valerate 2,3,7-Trimethyloctane 2,3,5-Trimethyldecane 2-Phenylpropenal 2,2,8-Trimethyldecane 1-Propylcyclohexane 2,5,5-Trimethyl-1-hexene 2,6,8-Trimethyldecane Trifluoroacetyl-isopulegol n-Tetradecanea n-Hexadecanea 1,5,5,8-Tetramethyl-3,7-cycloundecadien-1-ol 2,7,10-Terimethyldodecane 2,6,11-Trimethyldodecane 2,6-Dimethylheptadecane
13.02 13.35 13.61 13.74 14.52 15.34 15.86 15.94 17.74 18.79 18.93 19.11 19.22 19.42 19.60 20.17 21.28 21.29 22.25
1-Methylene-1H-indene 1,2-Benzisothiazole Azulenea Hexyl n-valerate 4,7-Dimethylundecane 2,3,7-Trimethyloctane 2,3,5-Trimethyldecane 2-Phenylpropenal 2-Methyl-5-propylnonane 2,5,5-Trimethyl-1-hexene 3,7-Dimethylundecane Tridecyliodide 3-Methylpentadecanea n-Tetradecanea n-Hexadecanea 4-Isopropyl-1,6-dimethylnaphthalene 2,2,4,4,6,8,8-Heptamethylnonane 7-Methylpentadecane 2,6-Dimethylheptadecane
13.02 13.46 13.60 15.34 15.94 16.10 17.11 18.79 18.84 19.42 19.51 20.41 20.67 20.79 20.92 21.29 22.25
1-Methylene-1H-indene 4-Methylnonanea Azulenea 2,3,7-Trimethyloctane 2-Phenylpropenal 2,3,4,4a,5,6,7,8-Octahydro-1,1,4a,7-tetramethyl-1H-benzocyclohepten-7-ol 2,2,8-Trimethyldecane 2,5,5-Trimethyl-1-hexene 1-Cyclohexyloctane n-Tetradecanea 5,6,6-Trimethylundeca-3,4-diene-2,10-dione 2,7,10-Trimethyldodecane 5-Propyltridecane 2,4-ditert-butyl-6-methylphenola 2,6,11-Trimethyldodecane 7-Methylpentadecane 2,6-Dimethylheptadecane
(min)
Compounds
RA (%) FS0
M. anisopliae (406)
M. anisopliae (EBCL 02049)
FS1
FS2
8.51
17.42
12.13
1.05 37.26
5.42 8.14
4.37 20.28
61.22 0.53 2.99 1.06
6.43 2.24 8.32 2.63
1.62 6.65 1.99 14.05 2.24 42.76 4.32
4.85
21.54
8.42 5.01 8.22 10.07 14.48 9.20
2.92 14.3
69.74
19.23
0.88
2.75 6.09 5.28
10.79 36.82
42.43 7.40 21.2 0.68 2.32 1.75 2.89 0.76 1.68
3.81
1.38
6.37
12.00
12.97
35.96 47.42 0.20
48.06 21.08 0.62
1.36
1.53 4.40
1.43 81.03 2.71 5.68
2.31
3.19 1.16
Rt = Retention time; RA = Relative area (peak area relative to total peak area); FS0 = In vitro culture (fungal strains successively cultured on PDA for 24 days); FS1 = 1st In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days as described in Section 2); FS2 = 2nd In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days). a Compounds identified by authentic standards.
but very little is known about the underlying mechanisms of virulence. The chromatographic fingerprints of the FS1 and FS2 cultures of M. anisopliae (02049) changed drastically to a profile with a straight-chain alkane (n-tetradecane) and 5,6,6-trimethylundeca3,4-diene-2,10-dione as the major compounds. Comparatively, the VOC profile of a less virulent inocula of the FS0 culture lacked these hydrocarbons and mainly comprised 2,3,4,4a,5,6,7,8-Octahydro-1,1,4a,7-tetramethyl-1H-benzocyclohepten-7-ol, alkenes, branched and cyclic alkanes. On the other hand, the volatile pro-
files of restored cultures of FS1 and FS2 of B. bassiana contained n-tetradecane, while the profile of the FS0 culture lacked this hydrocarbon and contained n-hexadecane. On the whole, all of the assays indicated the significant superiority of M. anisopliae over B. bassiana in terms of effectiveness in controlling C. formosanus workers, which is consistent with previous work reported by Jones et al. (1996) and Delate et al. (1995). The low LT50 values might be due to the production of 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane. These compounds may cause modifications in termite foraging behavior; if
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Fig. 1. Main fractions of volatile compounds Linear alkanes, branched-alkanes, Cyclic alkanes, Alkenes, Alcohols, Esters, Aldehydes, Ketones, Others (percentage of total peak areas) extracted from (a) B. bassiana, (b) M. anisopliae (406), (c) M. anisopliae (EBCL 02049) cultures [FS0 = In vitro culture (fungal strains successively cultured on PDA for 24 day); FS1 = 1st In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days); FS2 = 2nd In vivo culture (fungal strains cultivated from C. formosanus workers and then incubated on PDA for 24 days)]; using HS-SPME/GC–MS.
so, it becomes reasonable to hypothesize a new biological role for VOCs as a repellent to termites that would otherwise destroy the colony by natural fungal epizootics. Certainly, this concept is worthy of further study by directly testing the foraging behavior of termites upon direct exposure to fungal cultures. On the basis of the above findings, we may conclude that the difference in virulence may arise because of the change in the hydrocarbon profile. Normally, the VOC profiles of the virulent
inocula of the studied strains comprised mainly 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane. When the percentage of branched-alkanes in the volatile profile increased, the virulence decreased. However, further research is necessary to elucidate the mechanism responsible for this decrease in virulence. The incorporation of these prominent hydrocarbons into the growth media in order to observe their effects on the infectivity of entomopathogenic fungi will be the logical
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extension of this work in order to suggest a possible approach for mycoinsecticide improvement.
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