The role of diamondback moth cuticle surface compounds in pre-penetration growth of the entomopathogen Isaria fumosoroseus

Journal of Basic Microbiology 2010, 50, 411–419 411

© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

The role of diamondback moth cuticle surface compounds in pre-penetration growth of the entomopathogen Isaria fumosoroseus

Shaukat Ali, Zhen Huang1 and Shunxiang Ren

Engineering Research Center of Biological Control, Ministry of Education, College of Natural Resource and Environment, South China Agricultural University. Wushan Road, Guangzhou City, P.R. China, 510642

Transcript patterns elicited in response to hosts can reveal how fungi recognize suitable hosts and the mechanisms involved in pathogenicity. These patterns could be fashioned by recognition of host-specific topographical features or by chemical components displayed or released by the host. We investigated this in three isolates of Isaria fumosoroseus which has shown considerable pathogenic ability against Plutella xylostella. Conidia inoculated to hind wings had the highest germination ability while the conidia inoculated to head regions showed the lowest germination. Conidia applied to diamondback moth wings showed slightly lower rates of germination than those incubated on SDA. Although I. funosoroseus isolates germinated well on crude methanol extracts, only low levels of germination occurred on hexane and dichloromethane extracts. Similarly, poor germination was also observed on diamondback moth hind wing from which cuticular lipids were extracted by using methanol when compared to the untreated hind wings showing that simple polar compounds are required to stimulate germination before the fungus can make effective use of a complex mixture of non-polar lipids. Such studies could address the origin of intraspecies differences and correlate these differences with the underlying metabolic and biosynthetic differences that define different host ranges.

Keywords: Germination / Entomopathogenic fungi / Plutella xylostella / Cuticular compounds

Received: January 10, 2010; accepted: March 18, 2010

DOI 10.1002/jobm.201000014

Introduction1

Entomopathogenic fungi display different strategies in their attachment to insect cuticle which forms the first formidable barrier to pathogens and fungal spores have to pass through certain discrete stages before breaching the insect cuticle. According to Fragues et al. [1], the adhesion can be divided into three successive stages: (1) adsorption (passive phenomenon), (2) consolidation of the attachment, and (3) germination and growth of the fungus on the cuticular surface until penetration (active

1 Zhen Huang is the joint first author Correspondence: Shunxiang Ren, Engineering Research Center of Biological Control, Ministry of Education, College of Natural Resource and Environment, South China Agricultural University. Wushan Road, Guangzhou City, P.R. China, 510642 E-mail: [email protected] Phone: 0086-2085283508 Fax: 0086-2085280293

phenomenon). Adsorption of conidia to the cuticle is a nonspecific process mediated by electrostatic and hy-drophobic interactions [2, 3]. The attachment is followed by consolidation and germination, events which involve metabolic activity. The mechanisms for recognizing host-related triggers and the processes responsible for initiation of germination are likely involved in the ex-pression of virulence factors and parasitic specialization [4]. The hydrophobic conidia of many mycopathogens will bind in a nonspecific manner to the epicuticle sur-faces of both host and non host insects [3]. However, the production of penetrant germ tubes does not normally occur on non host insects. The germination process requires both an appropriate humidity regime and available nutrients for germ tube production [5]. The nature of the inductive triggers has not been determined, but while the protein and chitin composi-tion of insect procuticle appears similar in all insects,

412 S. Ali et al. Journal of Basic Microbiology 2010, 50, 411–419


the epicuticular components are extremely heteroge-neous, even within the same genus, and therefore have the potential to lead to different pathogen responses to particular insects [6, 7]. These epicuticular components of an insect cuticle may have both inhibitory and nutri-tional effects on the germination of conidia including, Beauveria bassiana and M. anisopliae; these effects could be due, in part, to the fatty acids present on the cuticle [8]. For Schistocerca gregaria (Forskal) these fatty acids occur at different levels with 1 ± 5% of the cuticle’s composition being free fatty acids for females and 4 ± 7% for males [9]. Some fatty acids may stimulate the initiation of germination and hyphal growth, while the others may inhibit both these processes [10]. Oleic and linoleic acids stimulate, while stearic and linolenic inhibit mycelial growth of Beauveria bassiana. Conidia of Nomuraea rileyi have been shown to rely completely on an exogenous supply of fatty acids and other substrates for germination [11]. Short chain fatty acids block the uptake of phosphate and thiamine in certain fungi, preventing the conidia from germinating [12]. Fungi have fatty acid carrier systems which effect selective uptake through fungal membranes according to carbon chain length also influencing which fatty acids inhibit and which promote germination [12]. A particular reason for focusing on Isaria fumosoroseus (Paecilomyces fumosoroseus designated as Isaria clade, Luangsa [13]) is the pathogenic ability of this mitosporic fungus to a wide variety of insects [14–16]. It can be easily cultured in vitro, and thus lend itself for use as microbial pesticides for several arthropod pests, includ-ing the diamondback moth, Plutella xylostella [14]. These applied interests would obviously benefit from an understanding of biochemical interactions between the pathogen and its hosts, particularly if genetic modifica-tions are to be designed to enhance pathogen attributes [17]. In the present work we investigated the growth behavior of I. fumosoroseus on diamondback moth cuti-cle by observing the attachment and germination pat-tern of the mitosporic fungal pathogen. The interaction between surface compounds from diamondback moth and I. fumosoroseus was also observed. Three isolates of I. fumosoroseus which has shown considerable pathogenic ability during the previous studies [14] were used dur-ing this study.

Materials and methods

Fungal strains For all assays, three fungal strains of entomopathogenic fungus I. fumosoroseus (IF-28-2, IF-32 and IF-49) originally

isolated from soil, maintained in tubes containing sabouraud dextrose agar (SDA) and deposited in the Engineering Research Center of Biological Control, South China Agricultural University, were cultured on potato dextrose agar (PDA) and incubated at 26 ± 2 °C for 10 d. Conidia were harvested with deion-ized water containing 0.03% Tween 80 and sieved through filter paper into sterile vials. Conidia were counted using a compound microscope in a hemocy-tometer (0.0625 m2; Fuchs-Rosenthal Merck Euro lab, Darmstadt, Germany) to calibrate a suspension of 1 × 106 conidia/ml.

Insect culture and wing preparation Adults of P. xylostella, were obtained from the stock cultures kept at 27 ± 1 °C, R.H. 75 ± 10% and a photo-period of 14:10 (L :D) in greenhouse of the Engineering Research Center of Biological Control, South China Agricultural University on Brassica campestris L., res-pectively. Plants were grown in plastic pots having a diameter of 15 cm. Slow release fertilizer (100 g, N:P:K = 13:7:15, Shenzhen Batian ecotypic engineer-ing Co., LTD. Xili Shenzhen China) was added as re-quired to maintain normal plant growth. Hind wings of diamondback moth were dissected away from live moths. Wings were surface sterilized in 5% sodium hypochlorite (5 min) and rinsed with 4 changes (5 min each) of sterile distilled water [4].

Extraction of epicuticular compounds Groups of 25 P. xyllostella adults were killed by freezing, freeze-dried, and hindwings were detached from the insect body. The wings were immersed in three to four fold excess of organic solvents (dichloromethane, hex-ane or methanol) and shaken at 50 rpm in rotary shaker for 10 min to extract cuticular lipids [18]. Wings were washed three times with each solvent. Extracts were transferred into airtight 2 ml glass vial and freeze dried to remove the solvent (dichloromethane, hexane or methanol). The extracts were then re-suspended in 1 ml each of the original solvent, di-chloromethane, hexane or methanol, and stored –20 °C until analyzed. Extraction of epicuticular compounds from the wings after 24 h of fungal growth was also carried out by using the above procedure after the removal of fun-gal spores from the wings. Fungal spores from the wing surface were removed by applying a controlled jet of distilled water for 20 s. The wings were observed under a flouresence microscope (Eclipse 80I Fluorescence Microscope, Nikon) to ensure that all the fungal sporess had been removed from the wings.

Journal of Basic Microbiology 2010, 50, 411–419 Growth of Isaria fumosoroseus on cuticular compounds 413


Wing bioassays For the wing bioassay the wings were used after the removal of different cuticular components as described above. Five wings from each extraction treatment were spread flat on 2% water agar plates and inoculated separately with 10 µl of 1 × 106 conidia/ml fungal sus-pension in dH2O, using a micro applicator and incu-bated at 27 °C and 80% humidity. Germination and appressorial counts were made on pieces cut from the center of the wing (to avoid edge effects), mounted on a microscope slide, stained with 0.1% cotton blue in lac-tophenol and covered with a cover slip. Only conidia on the membrane were assessed, not those on veins. Ger-mination was judged to have occurred when a germ tube was as long as it was broad. An appressorium was defined as an apical swelling on the germ tube of simi-lar size to a conidium.

In vitro assays A. Germination on different cuticular regions: P. xyllostella adults were killed by freezing, freeze-dried and the washed with dH2O, deproteinated, and delipidat- ed by the method used by Sosa-Gomez et al. [19]. The adults were immersed individually in 25 ml of a conid-ial suspension (1 × 106 conidia/ml) for 2 min and then rinsed three times in sterile distilled H2O to remove nonattached conidia. After being rinsed in H2O, the antennal, head, thorax, legs and wing regions were dissected from treated adults and incubated at 27 °C and 80% RH for 24 h. These cuticle samples were stained with lactophenol blue and examined at 500 X under fluorescence microscope (Eclipse 80I Fluores-cence Microscope, Nikon). A total of four microscopic fields of each region were examined to estimate the relative germination patterns of I. fumosoroseus to dia-mondback moth cuticle and the whole study was re-peated five times with freshly prepared fungal suspen-sion. B. Effect of nutrient sources on germination of I. fumosoroseus: Petri dishes separately having 9 ml of SDA (Sabouraud dextrose agar) and dH2O were inocu-lated with 10 µl of 1 × 106 conidia/ml fungal solution in dH2O, using a micro applicator and incubated without light at 27 °C and 80% RH. A glass cover slip was placed over a section of the each plate. and germination was assessed as described above. C. Effect of epicuticular extraction from wings on germination of I. fumosoroseus: Five wings from each extraction treatment were spread flat on 2% water agar plates and inoculated separately with 10 µl of 1 × 106 conidia/ml fungal suspension in distilled H2O, using a micro applicator and incubated at 27 °C and 80% hu-

midity. As a control treatment 10 µl of 1 × 106 conidia/ ml fungal suspension was applied to the untreated wing (wings without extraction) spread on 2% water agar plates. The whole study was repeated five times with freshly prepared fungal suspension. Germination counts were made as described above. D. Germination on different hexane, dichloro-methane and methanol extracts: Hexane, dichloro-methane or methanol diamondback moth wing ex-tracts (100 μl) were separately placed in the centre of a sterilized glass microscope cavity slide. The solvent was allowed to evaporate, and the slide was then inoculated with 10 µl of 1 × 106 conidia/ml fungal suspension. The preparation was incubated at 27 °C and 80% RH. Ger-mination and/or appressorium formation was assessed as described previously. E. Effect of different carbon and nitrogen sources on germination: Glucose, alanine and glycine solutions alone or in combination with each other were applied (1% v/v) in the centre of sterilized glass cavity micro-scope slide. The solvent was allowed to evaporate then all slides were inoculated with 10 µl of 1 × 106 co-nidia/ml fungal suspension. The preparation was incu-bated at 27 °C and 80% RH. Germination and/or ap-pressorium formation was assessed according to the procedure described previously.

Analysis of cuticular extracts Cuticular extracts were analyzed according to the method of Jarrold et al. [20] to determine the nature of different polar and non polar compounds present in these extracts.

Composition of methanol extract The different components of methanol extract apart from cuticular lipids were studied by the following procedures. Glucose oxidase assay as described by Xia et al. [21] was used to determine the amount of glucose present in the methanol extract. Protein estimation was carried out through Bradford assay using bovine albu-min serum (Sigma, USA) as a standard. Free amino acids and amino acids released from peptides by HCl hy-drolysis were analyzed according to the method of Spackman et al. [22].

Experimental design and statistical analysis Data regarding percentage germination after arcsine transformation were subjected to two way analysis of variance (ANOVA-2) between different treatments and isolates followed by the Tukey’s HSD test for mean comparison. SAS software (Version 8.02) was used for all statistical analysis [23].



Results

Germination (24 h post inoculation) of Isaria fumosoroseus isolates on different cuticular regions of diamondback moth The data about germination of conidia to various cuticle domains is presented in Fig. 1. After 24 h of fungal treatment, % germination of spores differed significantly among all cuticular regions (F4, 60 = 81.52, P < 0.01). Germination rate of spores from the different cuticular regions studied differed significantly among the isolates (F2, 60 = 52.41, P < 0.001). Similarly there was a significant interaction effect involving fungal strains and cuticular regions (F8, 60 = 11.05, P < 0.001). In all three isolates, conidia inoculated to wings had the highest germination ability of 93.35, 78.75 and 82.95% for IF28.2, IF32 and IF49, respectively. Conidia inocu-lated to head regions showed the lowest germination rate for each isolate tested (Fig. 1).

Effect of different nutrient sources on germination (24 h post inoculation) of Isaria fumosoroseus isolates The main effect of different nutrient sources (SDA as well as hind wings of diamondback moth) on % germi-nation of spores was significant (F2, 41 = 34.08, P < 0.01). Isaria fumosoroseus isolates also showed a significant on % spore germination (F2, 41 = 14.62, P < 0.001) as did the interaction of the two variables (F4, 41 = 1.59, P = 0.012). Germination rate of spores was highest for all three isolates in the SDA medium: 88.80, 81.26, and 79.31% for IF28.2, IF32, and IF49, respectively. Conidia inocu-

lated to dH2O showed the lowest germination rate for each isolate tested (Fig. 2).

Germination of Isaria fumosoroseus (24 h post inoculation) on diamondback moth wings extracted using solvent After 24 h of fungal treatment, % germination of spores differed significantly on sterile diamondback moth wings extracted with different solvents (F3, 48 = 26.07, P < 0.0001). % spore germination from sterile diamondback moth wings extracted with different solvents differed significantly in all isolates (F2, 288 = 26.51, P = 0.01). Similarly, a highly significant interac-tion effect between sterile diamondback moth wings extracted with different solvents and isolates was ob-served for % spore germination (F6, 48 = 11.25, P < 0.01). In all three isolates, conidia inoculated to wings treated with methanol had the lowest germination ability of 25.10, 22.84 and 16.06% for IF28.2, IF32 and IF49, respectively. Conidia inoculated to untreated wings showed the highest germination rate for each isolate tested (Fig. 3).

Germination of Isaria fumosoroseus (24 h post inoculation) on solvent wing extracts Germination rate of conidia differed significantly among all cuticular extracts as well as control treat-ments (F5, 72 = 41.50, P < 0.01). Germination rate of spores from the different cuticular extracts as well as control treatments studied differed significantly among the isolates (F2, 72 = 34.10, P < 0.001). Similarly there was a significant interaction effect on conidial germination

Figure 1. Germination of three Isaria fumosoroseus isolates on different cuticular regions of diamondback moth. Bars in the figure represent standard error of means. Legends with different letters are significantly different from each other (Tukey’s, <0.05).



Figure 2. Germination of three Isaria fumosoroseus isolates on different nutrient sources (24 h post inoculation). Bars in the figure represent standard error of means. Legends with different letters are significantly different from each other (Tukey’s, <0.05).

involving fungal strains and cuticular extracts (F10, 72 = 10.77, P < 0.001). The highest rates of germination (67.84, 61.74 and 43.42% for IF28.2, IF32 and IF49, re-spectively) were observed on methanol extract while the lowest rates of germination for each isolate tested were observed in control treatment with methanol solvent alone (Fig. 4).

Composition of cuticular extracts Composition analysis of cuticular extracts showed that hexane extract contained only hydrocarbons whereas polar extracts obtained through methanol extraction consisted of short chain hydrocarbons, methylated alkanes, long chain fatty acids and methyl ethyl esters (Table 1). Composition analysis methanol extract from

diamondback moth wings also showed some interesting results. Apart from polar lipids, Glucose (0.097 mM ± 0.003) and protein (0.07 mM ± 0.004) were also pre-sent. Alanine and glycine were observed to be the most dominant amino acids among free and released amino acids present in methanol extract (Table 2).

Effect of different carbon and nitrogen sources on germination of Isaria fumosoroseus (24 h post inoculation) Different carbon and nitrogen sources as well as their combination showed significantly different rates of germination when compared to the control (F6, 84 = 47.01, P = 0.018). Germination rate of I. fumosoroseus isolates varied significantly among different com-

Figure 3. Germination of three Isaria fumosoroseus isolates on diamondback moth wings extracted using solvent. Bars in the figure represent standard error of means. Legends with different letters are significantly different from each other (Tukey’s, <0.05).



Figure 4. Germination of three Isaria fumosoroseus isolates on solvent wing extracts. Bars in the figure represent standard error of means. Legends with different letters are significantly different from each other (Tukey’s, <0.05).

pounds and their combination within each isolate (F2, 84 = 22.05, P = 0.024). Interaction effects between different compounds and their combination and isolates also proved to be significantly different when compared for conidial germination per 300 conidia (F12, 84 = 7.54, P < 0.01). In all three isolates, conidia inoculated to glucose had the highest germination ability of 75, 69.17 and 65.45% for IF28.2, IF32 and IF49, respectively. Co-nidia inoculated to control showed the lowest germina-tion rate for each isolate tested (Fig. 5).

Discussion

One of the outstanding features of I. fumosoroseus is its ability to parasitize a wide range of different insect species. Many workers have characterized the host range of different isolates in attempts to characterize strains on the basis of pathogenecity [24]. The ability of different fungal isolates to attack some hosts and not others is related to the effects of different host compo-nents on adhesion and germination [25]. Therefore, this

Table 1. Composition of epicuticle extracts from diamondback moth wings.

Hexane extract Dichloromethane extract Methanol extract

Hydrocarbon (alkane) Pentacosane (C25), Heptacosane (C27), Nonacosane (C29), Tri- acontane (C30), Hentriacontane (C31)

Pentacosane (C25), Heptacosane (C27), Nonacosane (C29)

Pentadecane (C15), Heptadecane (C17), Tricosane (C23)

Methylated alkane Methylhexadecanoate (C16) 3-Methyl heptadecane (C17), 3-Methyl heneicosane (C21), 2-Methyl tricosane (C23)

Long-chain fatty acid Tetradecanoic acid (C14), Hexadecanoic acid (C16), 9,12 Octadecadienoic acid (C18),

Methyl-ethyl ester Methyloctadecanoate (C18) Ethyldecanoate (C12) Methylpenta-decanoate (C15), Ethyl-2-hydroxy-9-octadecenoate (C18)

Figures in parentheses (e.g. C25) indicate the number of carbon atoms in the molecule.



Table 2. Concentration of amino acids in methanol extracts from untreated diamondback moth wing epicuticle.

Amino acid Free amino acids (nmol/ml)

Amino acids post hydrolysis (nmol/ml)

Asparagine 2.8 12 Threonine 13 10.5 Serine 14 17 Glutamine 17 42 Proline 14 39 Glycine 84 121 Alanine 27 84 Cysteine 0.3 – Valine 6.9 17 Methionine – 1.5 Isoleucine 3.7 13 Leucine 3.9 17.4 Tyrosine 6.2 15.8 Phenylalanine 2.1 2.9 Histidine – – Lysine 5.8 7.3 Arginine 3.6 5.9

study was mainly focused on different host triggers which can be responsible to affect earliest stages of fungal infection like adhesion and germination. Previous research on cuticle-conidia binding deter-mined that hydrophobic interactions occurring be-tween the conidia wall and the epicuticle are respon-sible for the passive, nonspecific adhesion observed with hydrophobic conidia. Prior work with M. anisopliae demonstrated that conidia bind nonspecifically over the cuticle surface but can bedisplaced from smooth sclerite epicuticle more easily than from the epicuticle folds [26]. In several studies, M. anisopliae conidia have also shown an affinity to those regions containing setae

or spines [2] and to highly hydrophobic cuticle regions, such as the mosquito siphon tube [27]. The results of conidial binding studies performed with diamondback moth demonstrated that both topographical and chemical properties of the cuticle play important roles. The higher conidial densities were associated with the body regions containing numerous setae or spines. In certain cases, the conidia were trapped between adja-cent setae and depressions. However, the preferential binding to cuticle setae, which did not involve entrap-ment, suggested that the setae may have a unique sur-face chemistry [19]. Isaria fumosoroseus isolates germinated more quickly and more extensively on the readily available nutrient-rich medium than on diamondback moth cuticle (hind wings). Similarly, St. Leger et al. [28], showed that Metarhizium anisopliae isolate IMI 330189 produced ap-pressoria with equal facility on SDA and locust wings, whereas the isolate ARSEF 2575 was more fastidious. It required a hard hydrophobic surface and low concen-tration of a complex high molecular weight nitrogen containing compounds. These results also suggests that the fungus recognizes specific signals from the struc-tures or chemistry of the cuticle surface that vary with site, and that it behaves differently as a reaction to these signals and these stimuli are likely to be chemical rather than physical [4]. Germination on sterile diamondback moth wings extracted with methanol was significantly lower than on control wings for all the three isolates while extrac-tion with showed very low effect on germination com-pared to the control (Fig. 3). Likewise, the methanol extracts proved the most active inducers of fungal ger-

Figure 5. Germination of three Isaria fumosoroseus isolates on different carbon and nitrogen sources. Bars in the figure represent standard error of means. Legends with different letters are significantly different from each other (Tukey’s, <0.05).



mination when compared to other treatments and con-trol (Fig. 4). A possible reason of this phenomenon can be the composition of these extracts. Our results indi-cated that hexane extract contained only hydrocarbons whereas polar extracts obtained through methanol extraction consisted of short chain hydrocarbons, me-thylated alkanes, long chain fatty acids and methyl ethyl esters. Previous studies have also shown that polar insect cuticle lipid extracts stimulate germination [4, 11, 19, 29], though Lecuona et al. [8] found that the methanol extract from the whole-body cuticle of M. melolontha was inhibitory to germination. The synthetic fatty acids did inhibit conidial germination of P. fumoso-roseus, but not to the same extent as did the cuticular lipids, and they had no effect on B. bassiana germination [30]. It is possible that the fatty acids reduced the amount of moisture available to the spores on the membranes, rather than by a direct toxic effect. Napolitano and Juarez [31] found that mixtures of in-sect-derived hydrocarbons were more efficient than single authentic compounds in the isolates of M. ani-sopliae and Beauveria bassiana they studied. Interestingly, Wang and St. Leger [4] found that a non polar hexane extract of locust wings induced very low levels of ger-mination of the locust isolate M. anisopliae var. acridum ARSEF 324. However, supplementation of the lipoidal extract with yeast extract, raised germination to the high levels found on yeast extract alone. No discontinu-ity was found between the properties of whole metha-nol extracts and authentic polar constituents. The crude methanol extracts stimulated high levels of ger-mination as did components, e.g. glucose, amino acids (though only in combination with glucose). It is not clear where they came from in the first place however, such components were also found by Woods and Grula [5] in a polar extract from the cuticle of the lepidop-teran Heliothis zea and the amino acid composition of the extract was similar before and after proteolytic hydrolysis of lepidopterous cuticle. Glucose was the most effective chemical to support the germination. Amino acids like alanine and glycine by themselves showed a poor effect although in combi-nation with glucose statistically higher germination rates were observed but these mixtures were less effec-tive than glucose itself (Fig. 5). These differences in germination may have been due to the lack of a com-plex mix of nutrients, and may suggest that the carbon source is a more effective trigger for germination than the nitrogen source. Similar results were also observed by Jarrold et al. [19] for M. anisopliae. Germination and germ tube extension of M. anisopliae var. anisopliae ARSEF 2575 [32] and B. bassiana [5] were supported by

glucose and/or amino acids (particularly alanine and glycine). In conclusion, we have demonstrated that at least three different signals affect the germination of I. fumo-soroseus on diamondback moth: a hydrophobic surface, a polar cuticle fraction from an appropriate host and nutrient levels. The cuticle fraction extracted through methanol treatment, which contained glucose, amino acids and peptides in addition to the short chain hydro-carbons, methylated alkanes, long chain fatty acids and methyl ethyl esters, were strong promoters of germina-tion. Such studies could address the origin of intraspe-cies differences and correlate these differences with the underlying metabolic and biosynthetic differences that define different host ranges, identify the mechanisms by which novel pathogens emerge with different host ranges and identify targets for restricting or broaden-ing host ranges.

Acknowledgement

This research was funded by grants from the Ele- venth five year forest support program of China (No. 2006BAD08A1903).

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